<?xml version="1.0" encoding="US-ASCII" standalone="no"?>
    <!-- Field: Doc-Info; Name: Generator; Value: GoFiler Complete; Version: 5.25b -->
    <!-- Field: Doc-Info; Name: VendorURI; Value: https://www.novaworks.com -->
    <!-- Field: Doc-Info; Name: Status; Value: 0x00000000 -->
<link:linkbase xmlns:xsi="http://www.w3.org/2001/XMLSchema-instance" xmlns:xlink="http://www.w3.org/1999/xlink" xmlns:link="http://www.xbrl.org/2003/linkbase" xmlns:xbrldt="http://xbrl.org/2005/xbrldt" xmlns:xbrli="http://www.xbrl.org/2003/instance" xsi:schemaLocation="http://www.xbrl.org/2003/linkbase http://www.xbrl.org/2003/xbrl-linkbase-2003-12-31.xsd">
    <link:roleRef roleURI="http://n2off.com/role/Cover" xlink:href="nito-20241231.xsd#Cover" xlink:type="simple" />
    <link:roleRef roleURI="http://n2off.com/role/BalanceSheets" xlink:href="nito-20241231.xsd#BalanceSheets" xlink:type="simple" />
    <link:roleRef roleURI="http://n2off.com/role/BalanceSheetsParenthetical" xlink:href="nito-20241231.xsd#BalanceSheetsParenthetical" xlink:type="simple" />
    <link:roleRef roleURI="http://n2off.com/role/StatementsOfComprehensiveLoss" xlink:href="nito-20241231.xsd#StatementsOfComprehensiveLoss" xlink:type="simple" />
    <link:roleRef roleURI="http://n2off.com/role/StatementsOfChangesInStockholdersEquity" xlink:href="nito-20241231.xsd#StatementsOfChangesInStockholdersEquity" xlink:type="simple" />
    <link:roleRef roleURI="http://n2off.com/role/StatementsOfCashFlows" xlink:href="nito-20241231.xsd#StatementsOfCashFlows" xlink:type="simple" />
    <link:roleRef roleURI="http://xbrl.sec.gov/cyd/role/CybersecurityRiskManagementAndStrategyDisclosure" xlink:href="https://xbrl.sec.gov/cyd/2024/cyd-2024.xsd#CybersecurityRiskManagementAndStrategyDisclosure" xlink:type="simple" />
    <link:roleRef roleURI="http://xbrl.sec.gov/cyd/role/MaterialCybersecurityIncidentDisclosure" xlink:href="https://xbrl.sec.gov/cyd/2024/cyd-2024.xsd#MaterialCybersecurityIncidentDisclosure" xlink:type="simple" />
    <link:roleRef roleURI="http://n2off.com/role/General" xlink:href="nito-20241231.xsd#General" xlink:type="simple" />
    <link:roleRef roleURI="http://n2off.com/role/SignificantAccountingPolicies" xlink:href="nito-20241231.xsd#SignificantAccountingPolicies" xlink:type="simple" />
    <link:roleRef roleURI="http://n2off.com/role/OtherCurrentAssets" xlink:href="nito-20241231.xsd#OtherCurrentAssets" xlink:type="simple" />
    <link:roleRef roleURI="http://n2off.com/role/MitocarexTransaction" xlink:href="nito-20241231.xsd#MitocarexTransaction" xlink:type="simple" />
    <link:roleRef roleURI="http://n2off.com/role/PropertyAndEquipmentNet" xlink:href="nito-20241231.xsd#PropertyAndEquipmentNet" xlink:type="simple" />
    <link:roleRef roleURI="http://n2off.com/role/InvestmentInNonconsolidatedAffiliate" xlink:href="nito-20241231.xsd#InvestmentInNonconsolidatedAffiliate" xlink:type="simple" />
    <link:roleRef roleURI="http://n2off.com/role/InvestmentAndLoanToSolterra" xlink:href="nito-20241231.xsd#InvestmentAndLoanToSolterra" xlink:type="simple" />
    <link:roleRef roleURI="http://n2off.com/role/SolarPhotovolaicJointVentureProject" xlink:href="nito-20241231.xsd#SolarPhotovolaicJointVentureProject" xlink:type="simple" />
    <link:roleRef roleURI="http://n2off.com/role/ConsolidatedEntity" xlink:href="nito-20241231.xsd#ConsolidatedEntity" xlink:type="simple" />
    <link:roleRef roleURI="http://n2off.com/role/OtherLiabilities" xlink:href="nito-20241231.xsd#OtherLiabilities" xlink:type="simple" />
    <link:roleRef roleURI="http://n2off.com/role/CommitmentAndContingentLiabilities" xlink:href="nito-20241231.xsd#CommitmentAndContingentLiabilities" xlink:type="simple" />
    <link:roleRef roleURI="http://n2off.com/role/Leases" xlink:href="nito-20241231.xsd#Leases" xlink:type="simple" />
    <link:roleRef roleURI="http://n2off.com/role/StockholdersEquity" xlink:href="nito-20241231.xsd#StockholdersEquity" xlink:type="simple" />
    <link:roleRef roleURI="http://n2off.com/role/StockOptions" xlink:href="nito-20241231.xsd#StockOptions" xlink:type="simple" />
    <link:roleRef roleURI="http://n2off.com/role/CostOfSales" xlink:href="nito-20241231.xsd#CostOfSales" xlink:type="simple" />
    <link:roleRef roleURI="http://n2off.com/role/ResearchAndDevelopmentExpenses" xlink:href="nito-20241231.xsd#ResearchAndDevelopmentExpenses" xlink:type="simple" />
    <link:roleRef roleURI="http://n2off.com/role/SellingAndMarketingExpenses" xlink:href="nito-20241231.xsd#SellingAndMarketingExpenses" xlink:type="simple" />
    <link:roleRef roleURI="http://n2off.com/role/GeneralAndAdministrativeExpenses" xlink:href="nito-20241231.xsd#GeneralAndAdministrativeExpenses" xlink:type="simple" />
    <link:roleRef roleURI="http://n2off.com/role/FinancingExpensesIncomeNet" xlink:href="nito-20241231.xsd#FinancingExpensesIncomeNet" xlink:type="simple" />
    <link:roleRef roleURI="http://n2off.com/role/IncomeTax" xlink:href="nito-20241231.xsd#IncomeTax" xlink:type="simple" />
    <link:roleRef roleURI="http://n2off.com/role/LossPerShare" xlink:href="nito-20241231.xsd#LossPerShare" xlink:type="simple" />
    <link:roleRef roleURI="http://n2off.com/role/RelatedParties" xlink:href="nito-20241231.xsd#RelatedParties" xlink:type="simple" />
    <link:roleRef roleURI="http://n2off.com/role/SegmentReporting" xlink:href="nito-20241231.xsd#SegmentReporting" xlink:type="simple" />
    <link:roleRef roleURI="http://n2off.com/role/SubsequentEvents" xlink:href="nito-20241231.xsd#SubsequentEvents" xlink:type="simple" />
    <link:roleRef roleURI="http://n2off.com/role/SignificantAccountingPoliciesPolicies" xlink:href="nito-20241231.xsd#SignificantAccountingPoliciesPolicies" xlink:type="simple" />
    <link:roleRef roleURI="http://n2off.com/role/SignificantAccountingPoliciesTables" xlink:href="nito-20241231.xsd#SignificantAccountingPoliciesTables" xlink:type="simple" />
    <link:roleRef roleURI="http://n2off.com/role/OtherCurrentAssetsTables" xlink:href="nito-20241231.xsd#OtherCurrentAssetsTables" xlink:type="simple" />
    <link:roleRef roleURI="http://n2off.com/role/PropertyAndEquipmentNetTables" xlink:href="nito-20241231.xsd#PropertyAndEquipmentNetTables" xlink:type="simple" />
    <link:roleRef roleURI="http://n2off.com/role/InvestmentInNonconsolidatedAffiliateTables" xlink:href="nito-20241231.xsd#InvestmentInNonconsolidatedAffiliateTables" xlink:type="simple" />
    <link:roleRef roleURI="http://n2off.com/role/OtherLiabilitiesTables" xlink:href="nito-20241231.xsd#OtherLiabilitiesTables" xlink:type="simple" />
    <link:roleRef roleURI="http://n2off.com/role/CommitmentAndContingentLiabilitiesTables" xlink:href="nito-20241231.xsd#CommitmentAndContingentLiabilitiesTables" xlink:type="simple" />
    <link:roleRef roleURI="http://n2off.com/role/LeasesTables" xlink:href="nito-20241231.xsd#LeasesTables" xlink:type="simple" />
    <link:roleRef roleURI="http://n2off.com/role/StockOptionsTables" xlink:href="nito-20241231.xsd#StockOptionsTables" xlink:type="simple" />
    <link:roleRef roleURI="http://n2off.com/role/CostOfSalesTables" xlink:href="nito-20241231.xsd#CostOfSalesTables" xlink:type="simple" />
    <link:roleRef roleURI="http://n2off.com/role/ResearchAndDevelopmentExpensesTables" xlink:href="nito-20241231.xsd#ResearchAndDevelopmentExpensesTables" xlink:type="simple" />
    <link:roleRef roleURI="http://n2off.com/role/SellingAndMarketingExpensesTables" xlink:href="nito-20241231.xsd#SellingAndMarketingExpensesTables" xlink:type="simple" />
    <link:roleRef roleURI="http://n2off.com/role/GeneralAndAdministrativeExpensesTables" xlink:href="nito-20241231.xsd#GeneralAndAdministrativeExpensesTables" xlink:type="simple" />
    <link:roleRef roleURI="http://n2off.com/role/FinancingExpensesIncomeNetTables" xlink:href="nito-20241231.xsd#FinancingExpensesIncomeNetTables" xlink:type="simple" />
    <link:roleRef roleURI="http://n2off.com/role/IncomeTaxTables" xlink:href="nito-20241231.xsd#IncomeTaxTables" xlink:type="simple" />
    <link:roleRef roleURI="http://n2off.com/role/LossPerShareTables" xlink:href="nito-20241231.xsd#LossPerShareTables" xlink:type="simple" />
    <link:roleRef roleURI="http://n2off.com/role/RelatedPartiesTables" xlink:href="nito-20241231.xsd#RelatedPartiesTables" xlink:type="simple" />
    <link:roleRef roleURI="http://n2off.com/role/SegmentReportingTables" xlink:href="nito-20241231.xsd#SegmentReportingTables" xlink:type="simple" />
    <link:roleRef roleURI="http://n2off.com/role/GeneralDetailsNarrative" xlink:href="nito-20241231.xsd#GeneralDetailsNarrative" xlink:type="simple" />
    <link:roleRef roleURI="http://n2off.com/role/ScheduleOfPropertyPlantAndEquipmentDepreciationRatesDetails" xlink:href="nito-20241231.xsd#ScheduleOfPropertyPlantAndEquipmentDepreciationRatesDetails" xlink:type="simple" />
    <link:roleRef roleURI="http://n2off.com/role/ScheduleOfFairValueAssetsOnRecurringBasisDetails" xlink:href="nito-20241231.xsd#ScheduleOfFairValueAssetsOnRecurringBasisDetails" xlink:type="simple" />
    <link:roleRef roleURI="http://n2off.com/role/ScheduleOfChangesInFairValueOfAssetsDetails" xlink:href="nito-20241231.xsd#ScheduleOfChangesInFairValueOfAssetsDetails" xlink:type="simple" />
    <link:roleRef roleURI="http://n2off.com/role/SignificantAccountingPoliciesDetailsNarrative" xlink:href="nito-20241231.xsd#SignificantAccountingPoliciesDetailsNarrative" xlink:type="simple" />
    <link:roleRef roleURI="http://n2off.com/role/ScheduleOfOtherCurrentAssetsDetails" xlink:href="nito-20241231.xsd#ScheduleOfOtherCurrentAssetsDetails" xlink:type="simple" />
    <link:roleRef roleURI="http://n2off.com/role/MitocarexTransactionDetailsNarrative" xlink:href="nito-20241231.xsd#MitocarexTransactionDetailsNarrative" xlink:type="simple" />
    <link:roleRef roleURI="http://n2off.com/role/ScheduleOfPropertyPlantAndEquipmentDetails" xlink:href="nito-20241231.xsd#ScheduleOfPropertyPlantAndEquipmentDetails" xlink:type="simple" />
    <link:roleRef roleURI="http://n2off.com/role/PropertyAndEquipmentNetDetailsNarrative" xlink:href="nito-20241231.xsd#PropertyAndEquipmentNetDetailsNarrative" xlink:type="simple" />
    <link:roleRef roleURI="http://n2off.com/role/ScheduleOfFairValueConversionDetails" xlink:href="nito-20241231.xsd#ScheduleOfFairValueConversionDetails" xlink:type="simple" />
    <link:roleRef roleURI="http://n2off.com/role/ScheduleOfEquityInvestmentDetails" xlink:href="nito-20241231.xsd#ScheduleOfEquityInvestmentDetails" xlink:type="simple" />
    <link:roleRef roleURI="http://n2off.com/role/InvestmentInNonconsolidatedAffiliateDetailsNarrative" xlink:href="nito-20241231.xsd#InvestmentInNonconsolidatedAffiliateDetailsNarrative" xlink:type="simple" />
    <link:roleRef roleURI="http://n2off.com/role/InvestmentAndLoanToSolterraDetailsNarrative" xlink:href="nito-20241231.xsd#InvestmentAndLoanToSolterraDetailsNarrative" xlink:type="simple" />
    <link:roleRef roleURI="http://n2off.com/role/SolarPhotovolaicJointVentureProjectDetailsNarrative" xlink:href="nito-20241231.xsd#SolarPhotovolaicJointVentureProjectDetailsNarrative" xlink:type="simple" />
    <link:roleRef roleURI="http://n2off.com/role/ConsolidatedEntityDetailsNarrative" xlink:href="nito-20241231.xsd#ConsolidatedEntityDetailsNarrative" xlink:type="simple" />
    <link:roleRef roleURI="http://n2off.com/role/ScheduleOfOtherLiabilitiesDetails" xlink:href="nito-20241231.xsd#ScheduleOfOtherLiabilitiesDetails" xlink:type="simple" />
    <link:roleRef roleURI="http://n2off.com/role/ScheduleOfWarrantAssumptionsUsedDetails" xlink:href="nito-20241231.xsd#ScheduleOfWarrantAssumptionsUsedDetails" xlink:type="simple" />
    <link:roleRef roleURI="http://n2off.com/role/CommitmentAndContingentLiabilitiesDetailsNarrative" xlink:href="nito-20241231.xsd#CommitmentAndContingentLiabilitiesDetailsNarrative" xlink:type="simple" />
    <link:roleRef roleURI="http://n2off.com/role/ScheduleOfOperatingLeaseCostDetails" xlink:href="nito-20241231.xsd#ScheduleOfOperatingLeaseCostDetails" xlink:type="simple" />
    <link:roleRef roleURI="http://n2off.com/role/ScheduleOfSupplementalCashFlowInformationRelatedToOperatingLeaseDetails" xlink:href="nito-20241231.xsd#ScheduleOfSupplementalCashFlowInformationRelatedToOperatingLeaseDetails" xlink:type="simple" />
    <link:roleRef roleURI="http://n2off.com/role/ScheduleOfSupplementalBalanceSheetInformationRelatedToOperatingLeasesDetails" xlink:href="nito-20241231.xsd#ScheduleOfSupplementalBalanceSheetInformationRelatedToOperatingLeasesDetails" xlink:type="simple" />
    <link:roleRef roleURI="http://n2off.com/role/ScheduleOfMinimumLeasePaymentsUnderNon-CancelableLeasesDetails" xlink:href="nito-20241231.xsd#ScheduleOfMinimumLeasePaymentsUnderNon-CancelableLeasesDetails" xlink:type="simple" />
    <link:roleRef roleURI="http://n2off.com/role/LeasesDetailsNarrative" xlink:href="nito-20241231.xsd#LeasesDetailsNarrative" xlink:type="simple" />
    <link:roleRef roleURI="http://n2off.com/role/ScheduleOfTransactionsAndBalancesWithRelatedPartiesDetailsParenthetical" xlink:href="nito-20241231.xsd#ScheduleOfTransactionsAndBalancesWithRelatedPartiesDetailsParenthetical" xlink:type="simple" />
    <link:roleRef roleURI="http://n2off.com/role/StockholdersEquityDetailsNarrative" xlink:href="nito-20241231.xsd#StockholdersEquityDetailsNarrative" xlink:type="simple" />
    <link:roleRef roleURI="http://n2off.com/role/ScheduleOfEstimatedFairValueOfOptionsGrantedDetails" xlink:href="nito-20241231.xsd#ScheduleOfEstimatedFairValueOfOptionsGrantedDetails" xlink:type="simple" />
    <link:roleRef roleURI="http://n2off.com/role/ScheduleOfEstimatedFairValueOfOptionsGrantedDetailsParenthetical" xlink:href="nito-20241231.xsd#ScheduleOfEstimatedFairValueOfOptionsGrantedDetailsParenthetical" xlink:type="simple" />
    <link:roleRef roleURI="http://n2off.com/role/ScheduleOfStockOptionActivityDetails" xlink:href="nito-20241231.xsd#ScheduleOfStockOptionActivityDetails" xlink:type="simple" />
    <link:roleRef roleURI="http://n2off.com/role/StockOptionsDetailsNarrative" xlink:href="nito-20241231.xsd#StockOptionsDetailsNarrative" xlink:type="simple" />
    <link:roleRef roleURI="http://n2off.com/role/ScheduleOfCostOfSalesDetails" xlink:href="nito-20241231.xsd#ScheduleOfCostOfSalesDetails" xlink:type="simple" />
    <link:roleRef roleURI="http://n2off.com/role/ScheduleOfResearchAndDevelopmentExpensesDetails" xlink:href="nito-20241231.xsd#ScheduleOfResearchAndDevelopmentExpensesDetails" xlink:type="simple" />
    <link:roleRef roleURI="http://n2off.com/role/ScheduleOfSellingAndMarketingExpensesDetails" xlink:href="nito-20241231.xsd#ScheduleOfSellingAndMarketingExpensesDetails" xlink:type="simple" />
    <link:roleRef roleURI="http://n2off.com/role/ScheduleOfGeneralAndAdministrativeExpensesDetails" xlink:href="nito-20241231.xsd#ScheduleOfGeneralAndAdministrativeExpensesDetails" xlink:type="simple" />
    <link:roleRef roleURI="http://n2off.com/role/ScheduleOfFinancingExpensesNetDetails" xlink:href="nito-20241231.xsd#ScheduleOfFinancingExpensesNetDetails" xlink:type="simple" />
    <link:roleRef roleURI="http://n2off.com/role/ScheduleOfIncomeTaxExpensesDetails" xlink:href="nito-20241231.xsd#ScheduleOfIncomeTaxExpensesDetails" xlink:type="simple" />
    <link:roleRef roleURI="http://n2off.com/role/ScheduleOfDeferredTaxAssetsAndLiabilitiesDetails" xlink:href="nito-20241231.xsd#ScheduleOfDeferredTaxAssetsAndLiabilitiesDetails" xlink:type="simple" />
    <link:roleRef roleURI="http://n2off.com/role/IncomeTaxDetailsNarrative" xlink:href="nito-20241231.xsd#IncomeTaxDetailsNarrative" xlink:type="simple" />
    <link:roleRef roleURI="http://n2off.com/role/ScheduleOfBasicAndDilutedLossPerShareDetails" xlink:href="nito-20241231.xsd#ScheduleOfBasicAndDilutedLossPerShareDetails" xlink:type="simple" />
    <link:roleRef roleURI="http://n2off.com/role/ScheduleOfTransactionsAndBalancesWithRelatedPartiesDetails" xlink:href="nito-20241231.xsd#ScheduleOfTransactionsAndBalancesWithRelatedPartiesDetails" xlink:type="simple" />
    <link:roleRef roleURI="http://n2off.com/role/RelatedPartiesDetailsNarrative" xlink:href="nito-20241231.xsd#RelatedPartiesDetailsNarrative" xlink:type="simple" />
    <link:roleRef roleURI="http://n2off.com/role/ScheduleOfInformationRelatedToOperationsOfReportableOperatingSegmentsDetails" xlink:href="nito-20241231.xsd#ScheduleOfInformationRelatedToOperationsOfReportableOperatingSegmentsDetails" xlink:type="simple" />
    <link:roleRef roleURI="http://n2off.com/role/ScheduleOfInformationOnSalesByGeographicDistributionDetails" xlink:href="nito-20241231.xsd#ScheduleOfInformationOnSalesByGeographicDistributionDetails" xlink:type="simple" />
    <link:roleRef roleURI="http://n2off.com/role/ScheduleOfSalesToCustomersDetails" xlink:href="nito-20241231.xsd#ScheduleOfSalesToCustomersDetails" xlink:type="simple" />
    <link:roleRef roleURI="http://n2off.com/role/ScheduleOfInformationOnLongLivedAssetsDetails" xlink:href="nito-20241231.xsd#ScheduleOfInformationOnLongLivedAssetsDetails" xlink:type="simple" />
    <link:roleRef roleURI="http://n2off.com/role/SegmentReportingDetailsNarrative" xlink:href="nito-20241231.xsd#SegmentReportingDetailsNarrative" xlink:type="simple" />
    <link:roleRef roleURI="http://n2off.com/role/SubsequentEventsDetailsNarrative" xlink:href="nito-20241231.xsd#SubsequentEventsDetailsNarrative" xlink:type="simple" />
    <link:roleRef roleURI="http://xbrl.sec.gov/ecd/role/PvpDisclosureWithAdditionalMeasures" xlink:href="https://xbrl.sec.gov/ecd/2024/ecd-2024.xsd#PvpDisclosureWithAdditionalMeasures" xlink:type="simple" />
    <link:roleRef roleURI="http://xbrl.sec.gov/ecd/role/PvpTable" xlink:href="https://xbrl.sec.gov/ecd/2024/ecd-2024.xsd#PvpTable" xlink:type="simple" />
    <link:roleRef roleURI="http://xbrl.sec.gov/ecd/role/PvpAdjustmentsToCompensation" xlink:href="https://xbrl.sec.gov/ecd/2024/ecd-2024.xsd#PvpAdjustmentsToCompensation" xlink:type="simple" />
    <link:roleRef roleURI="http://xbrl.sec.gov/ecd/role/TabularListsByExecutiveCategory" xlink:href="https://xbrl.sec.gov/ecd/2024/ecd-2024.xsd#TabularListsByExecutiveCategory" xlink:type="simple" />
    <link:roleRef roleURI="http://xbrl.sec.gov/ecd/role/TabularListMeasures" xlink:href="https://xbrl.sec.gov/ecd/2024/ecd-2024.xsd#TabularListMeasures" xlink:type="simple" />
    <link:roleRef roleURI="http://xbrl.sec.gov/ecd/role/PvpMeasures" xlink:href="https://xbrl.sec.gov/ecd/2024/ecd-2024.xsd#PvpMeasures" xlink:type="simple" />
    <link:roleRef roleURI="http://xbrl.sec.gov/ecd/role/PvpExecutiveCategoriesOnly" xlink:href="https://xbrl.sec.gov/ecd/2024/ecd-2024.xsd#PvpExecutiveCategoriesOnly" xlink:type="simple" />
    <link:roleRef roleURI="http://xbrl.sec.gov/ecd/role/PvpAdjustmentsOnly" xlink:href="https://xbrl.sec.gov/ecd/2024/ecd-2024.xsd#PvpAdjustmentsOnly" xlink:type="simple" />
    <link:roleRef roleURI="http://xbrl.sec.gov/ecd/role/IndividualsOnly" xlink:href="https://xbrl.sec.gov/ecd/2024/ecd-2024.xsd#IndividualsOnly" xlink:type="simple" />
    <link:roleRef roleURI="http://xbrl.sec.gov/ecd/AwardTypeOnly" xlink:href="https://xbrl.sec.gov/ecd/2024/ecd-2024.xsd#AwardTypeOnly" xlink:type="simple" />
    <link:roleRef roleURI="http://xbrl.sec.gov/ecd/role/TradingArrangementsOnly" xlink:href="https://xbrl.sec.gov/ecd/2024/ecd-2024.xsd#TradingArrangmentsOnly" xlink:type="simple" />
    <link:roleRef roleURI="http://xbrl.sec.gov/ecd/role/ForgoneRecoveries" xlink:href="https://xbrl.sec.gov/ecd/2024/ecd-2024.xsd#ForgoneRecoveries" xlink:type="simple" />
    <link:roleRef roleURI="http://xbrl.sec.gov/ecd/role/OutstandingRecoveries" xlink:href="https://xbrl.sec.gov/ecd/2024/ecd-2024.xsd#OutstandingRecoveries" xlink:type="simple" />
    <link:roleRef roleURI="http://xbrl.sec.gov/ecd/role/AwardsCloseToMnpiDisc" xlink:href="https://xbrl.sec.gov/ecd/2024/ecd-2024.xsd#AwardsCloseToMnpiDisc" xlink:type="simple" />
    <link:roleRef roleURI="http://xbrl.sec.gov/ecd/role/InsiderTradingArrangementsByInd" xlink:href="https://xbrl.sec.gov/ecd/2024/ecd-2024.xsd#InsiderTradingArrangemenstByInd" xlink:type="simple" />
    <link:roleRef roleURI="http://xbrl.sec.gov/ecd/role/Defaults" xlink:href="https://xbrl.sec.gov/ecd/2024/ecd-2024.xsd#Defaults" xlink:type="simple" />
    <link:arcroleRef xlink:type="simple" xlink:href="http://www.xbrl.org/2005/xbrldt-2005.xsd#hypercube-dimension" arcroleURI="http://xbrl.org/int/dim/arcrole/hypercube-dimension" />
    <link:arcroleRef xlink:type="simple" xlink:href="http://www.xbrl.org/2005/xbrldt-2005.xsd#dimension-domain" arcroleURI="http://xbrl.org/int/dim/arcrole/dimension-domain" />
    <link:arcroleRef xlink:type="simple" xlink:href="http://www.xbrl.org/2005/xbrldt-2005.xsd#domain-member" arcroleURI="http://xbrl.org/int/dim/arcrole/domain-member" />
    <link:arcroleRef xlink:type="simple" xlink:href="http://www.xbrl.org/2005/xbrldt-2005.xsd#all" arcroleURI="http://xbrl.org/int/dim/arcrole/all" />
    <link:arcroleRef xlink:type="simple" xlink:href="http://www.xbrl.org/2005/xbrldt-2005.xsd#notAll" arcroleURI="http://xbrl.org/int/dim/arcrole/notAll" />
    <link:arcroleRef xlink:type="simple" xlink:href="http://www.xbrl.org/2005/xbrldt-2005.xsd#dimension-default" arcroleURI="http://xbrl.org/int/dim/arcrole/dimension-default" />
    <link:definitionLink xlink:type="extended" xlink:role="http://n2off.com/role/Cover" xlink:title="00000001 - Document - Cover" />
    <link:definitionLink xlink:type="extended" xlink:role="http://n2off.com/role/BalanceSheets" xlink:title="00000002 - Statement - Consolidated Balance Sheets" />
    <link:definitionLink xlink:type="extended" xlink:role="http://n2off.com/role/BalanceSheetsParenthetical" xlink:title="00000003 - Statement - Consolidated Balance Sheets (Parenthetical)" />
    <link:definitionLink xlink:type="extended" xlink:role="http://n2off.com/role/StatementsOfComprehensiveLoss" xlink:title="00000004 - Statement - Consolidated Statements of Comprehensive Loss" />
    <link:definitionLink xlink:type="extended" xlink:role="http://n2off.com/role/StatementsOfChangesInStockholdersEquity" xlink:title="00000005 - Statement - Statements of Changes In Stockholders&apos; Equity">
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_StatementLineItems" xlink:label="loc_us-gaapStatementLineItems" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_StatementTable" xlink:label="loc_us-gaapStatementTable" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xbrldt:contextElement="segment" xlink:from="loc_us-gaapStatementLineItems" xlink:to="loc_us-gaapStatementTable" xlink:type="arc" order="10" xbrldt:closed="true" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_StatementEquityComponentsAxis" xlink:label="loc_us-gaapStatementEquityComponentsAxis" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_us-gaapStatementTable" xlink:to="loc_us-gaapStatementEquityComponentsAxis" xlink:type="arc" order="1" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_EquityComponentDomain" xlink:label="loc_us-gaapEquityComponentDomain" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_us-gaapStatementEquityComponentsAxis" xlink:to="loc_us-gaapEquityComponentDomain" xlink:type="arc" order="20" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_EquityComponentDomain" xlink:label="loc_us-gaapEquityComponentDomain_10" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xbrldt:contextElement="segment" xlink:from="loc_us-gaapStatementEquityComponentsAxis" xlink:to="loc_us-gaapEquityComponentDomain_10" xlink:type="arc" order="30" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_CommonStockMember" xlink:label="loc_us-gaapCommonStockMember_0" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapEquityComponentDomain" xlink:to="loc_us-gaapCommonStockMember_0" xlink:type="arc" order="1" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_AdditionalPaidInCapitalMember" xlink:label="loc_us-gaapAdditionalPaidInCapitalMember_10" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapEquityComponentDomain" xlink:to="loc_us-gaapAdditionalPaidInCapitalMember_10" xlink:type="arc" order="11" />
      <link:loc xlink:type="locator" xlink:href="nito-20241231.xsd#NITO_ForeignCurrencyTranslationAdjustmentsMember" xlink:label="loc_NITOForeignCurrencyTranslationAdjustmentsMember_20" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapEquityComponentDomain" xlink:to="loc_NITOForeignCurrencyTranslationAdjustmentsMember_20" xlink:type="arc" order="21" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_RetainedEarningsMember" xlink:label="loc_us-gaapRetainedEarningsMember_30" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapEquityComponentDomain" xlink:to="loc_us-gaapRetainedEarningsMember_30" xlink:type="arc" order="31" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_ParentMember" xlink:label="loc_us-gaapParentMember_40" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapEquityComponentDomain" xlink:to="loc_us-gaapParentMember_40" xlink:type="arc" order="41" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_NoncontrollingInterestMember" xlink:label="loc_us-gaapNoncontrollingInterestMember_50" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapEquityComponentDomain" xlink:to="loc_us-gaapNoncontrollingInterestMember_50" xlink:type="arc" order="51" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_StockholdersEquityIncludingPortionAttributableToNoncontrollingInterest" xlink:label="loc_us-gaapStockholdersEquityIncludingPortionAttributableToNoncontrollingInterest_80" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapStatementLineItems" xlink:to="loc_us-gaapStockholdersEquityIncludingPortionAttributableToNoncontrollingInterest_80" xlink:type="arc" order="1" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_SharesOutstanding" xlink:label="loc_us-gaapSharesOutstanding_80" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapStatementLineItems" xlink:to="loc_us-gaapSharesOutstanding_80" xlink:type="arc" order="2" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_StockIssuedDuringPeriodValueConversionOfConvertibleSecurities" xlink:label="loc_us-gaapStockIssuedDuringPeriodValueConversionOfConvertibleSecurities_80" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapStatementLineItems" xlink:to="loc_us-gaapStockIssuedDuringPeriodValueConversionOfConvertibleSecurities_80" xlink:type="arc" order="3" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_StockIssuedDuringPeriodSharesConversionOfConvertibleSecurities" xlink:label="loc_us-gaapStockIssuedDuringPeriodSharesConversionOfConvertibleSecurities_80" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapStatementLineItems" xlink:to="loc_us-gaapStockIssuedDuringPeriodSharesConversionOfConvertibleSecurities_80" xlink:type="arc" order="4" />
      <link:loc xlink:type="locator" xlink:href="nito-20241231.xsd#NITO_IssuanceOfSharesForYaaranExchangeAgreementInValue" xlink:label="loc_NITOIssuanceOfSharesForYaaranExchangeAgreementInValue_80" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapStatementLineItems" xlink:to="loc_NITOIssuanceOfSharesForYaaranExchangeAgreementInValue_80" xlink:type="arc" order="5" />
      <link:loc xlink:type="locator" xlink:href="nito-20241231.xsd#NITO_IssuanceOfSharesForYaaranExchangeAgreementInShares" xlink:label="loc_NITOIssuanceOfSharesForYaaranExchangeAgreementInShares_80" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapStatementLineItems" xlink:to="loc_NITOIssuanceOfSharesForYaaranExchangeAgreementInShares_80" xlink:type="arc" order="6" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_StockIssuedDuringPeriodValueIssuedForServices" xlink:label="loc_us-gaapStockIssuedDuringPeriodValueIssuedForServices_80" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapStatementLineItems" xlink:to="loc_us-gaapStockIssuedDuringPeriodValueIssuedForServices_80" xlink:type="arc" order="7" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_StockIssuedDuringPeriodSharesIssuedForServices" xlink:label="loc_us-gaapStockIssuedDuringPeriodSharesIssuedForServices_80" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapStatementLineItems" xlink:to="loc_us-gaapStockIssuedDuringPeriodSharesIssuedForServices_80" xlink:type="arc" order="8" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_StockIssuedDuringPeriodValueShareBasedCompensation" xlink:label="loc_us-gaapStockIssuedDuringPeriodValueShareBasedCompensation_80" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapStatementLineItems" xlink:to="loc_us-gaapStockIssuedDuringPeriodValueShareBasedCompensation_80" xlink:type="arc" order="9" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_StockIssuedDuringPeriodValuePurchaseOfAssets" xlink:label="loc_us-gaapStockIssuedDuringPeriodValuePurchaseOfAssets_80" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapStatementLineItems" xlink:to="loc_us-gaapStockIssuedDuringPeriodValuePurchaseOfAssets_80" xlink:type="arc" order="10" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_StockIssuedDuringPeriodSharesPurchaseOfAssets" xlink:label="loc_us-gaapStockIssuedDuringPeriodSharesPurchaseOfAssets_80" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapStatementLineItems" xlink:to="loc_us-gaapStockIssuedDuringPeriodSharesPurchaseOfAssets_80" xlink:type="arc" order="11" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_StockIssuedDuringPeriodValueNewIssues" xlink:label="loc_us-gaapStockIssuedDuringPeriodValueNewIssues_80" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapStatementLineItems" xlink:to="loc_us-gaapStockIssuedDuringPeriodValueNewIssues_80" xlink:type="arc" order="12" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_StockIssuedDuringPeriodSharesNewIssues" xlink:label="loc_us-gaapStockIssuedDuringPeriodSharesNewIssues_80" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapStatementLineItems" xlink:to="loc_us-gaapStockIssuedDuringPeriodSharesNewIssues_80" xlink:type="arc" order="13" />
      <link:loc xlink:type="locator" xlink:href="nito-20241231.xsd#NITO_StockIssuedDuringPeriodValueTransactionsWithNoncontrollingInterests" xlink:label="loc_NITOStockIssuedDuringPeriodValueTransactionsWithNoncontrollingInterests_80" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapStatementLineItems" xlink:to="loc_NITOStockIssuedDuringPeriodValueTransactionsWithNoncontrollingInterests_80" xlink:type="arc" order="14" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_ProfitLoss" xlink:label="loc_us-gaapProfitLoss_80" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapStatementLineItems" xlink:to="loc_us-gaapProfitLoss_80" xlink:type="arc" order="15" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_StockIssuedDuringPeriodValueOther" xlink:label="loc_us-gaapStockIssuedDuringPeriodValueOther_80" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapStatementLineItems" xlink:to="loc_us-gaapStockIssuedDuringPeriodValueOther_80" xlink:type="arc" order="16" />
    </link:definitionLink>
    <link:definitionLink xlink:type="extended" xlink:role="http://n2off.com/role/StatementsOfCashFlows" xlink:title="00000006 - Statement - Consolidated Statements of Cash Flows" />
    <link:definitionLink xlink:type="extended" xlink:role="http://xbrl.sec.gov/cyd/role/CybersecurityRiskManagementAndStrategyDisclosure" xlink:title="00000012 - Disclosure - Cybersecurity Risk Management and Strategy Disclosure" />
    <link:definitionLink xlink:type="extended" xlink:role="http://xbrl.sec.gov/cyd/role/MaterialCybersecurityIncidentDisclosure" xlink:title="00000013 - Disclosure - Material Cybersecurity Incident Disclosure" />
    <link:definitionLink xlink:type="extended" xlink:role="http://n2off.com/role/General" xlink:title="999014 - Disclosure - GENERAL" />
    <link:definitionLink xlink:type="extended" xlink:role="http://n2off.com/role/SignificantAccountingPolicies" xlink:title="999015 - Disclosure - SIGNIFICANT ACCOUNTING POLICIES" />
    <link:definitionLink xlink:type="extended" xlink:role="http://n2off.com/role/OtherCurrentAssets" xlink:title="999016 - Disclosure - OTHER CURRENT ASSETS" />
    <link:definitionLink xlink:type="extended" xlink:role="http://n2off.com/role/MitocarexTransaction" xlink:title="999017 - Disclosure - MITOCAREX TRANSACTION" />
    <link:definitionLink xlink:type="extended" xlink:role="http://n2off.com/role/PropertyAndEquipmentNet" xlink:title="999018 - Disclosure - PROPERTY AND EQUIPMENT, NET" />
    <link:definitionLink xlink:type="extended" xlink:role="http://n2off.com/role/InvestmentInNonconsolidatedAffiliate" xlink:title="999019 - Disclosure - INVESTMENT IN NONCONSOLIDATED AFFILIATE" />
    <link:definitionLink xlink:type="extended" xlink:role="http://n2off.com/role/InvestmentAndLoanToSolterra" xlink:title="999020 - Disclosure - INVESTMENT AND LOAN TO SOLTERRA" />
    <link:definitionLink xlink:type="extended" xlink:role="http://n2off.com/role/SolarPhotovolaicJointVentureProject" xlink:title="999021 - Disclosure - SOLAR PHOTOVOLAIC JOINT VENTURE PROJECT" />
    <link:definitionLink xlink:type="extended" xlink:role="http://n2off.com/role/ConsolidatedEntity" xlink:title="999022 - Disclosure - A CONSOLIDATED ENTITY" />
    <link:definitionLink xlink:type="extended" xlink:role="http://n2off.com/role/OtherLiabilities" xlink:title="999023 - Disclosure - OTHER LIABILITIES" />
    <link:definitionLink xlink:type="extended" xlink:role="http://n2off.com/role/CommitmentAndContingentLiabilities" xlink:title="999024 - Disclosure - COMMITMENT AND CONTINGENT LIABILITIES" />
    <link:definitionLink xlink:type="extended" xlink:role="http://n2off.com/role/Leases" xlink:title="999025 - Disclosure - LEASES" />
    <link:definitionLink xlink:type="extended" xlink:role="http://n2off.com/role/StockholdersEquity" xlink:title="999026 - Disclosure - STOCKHOLDERS&#8217; EQUITY" />
    <link:definitionLink xlink:type="extended" xlink:role="http://n2off.com/role/StockOptions" xlink:title="999027 - Disclosure - STOCK OPTIONS" />
    <link:definitionLink xlink:type="extended" xlink:role="http://n2off.com/role/CostOfSales" xlink:title="999028 - Disclosure - COST OF SALES" />
    <link:definitionLink xlink:type="extended" xlink:role="http://n2off.com/role/ResearchAndDevelopmentExpenses" xlink:title="999029 - Disclosure - RESEARCH AND DEVELOPMENT EXPENSES" />
    <link:definitionLink xlink:type="extended" xlink:role="http://n2off.com/role/SellingAndMarketingExpenses" xlink:title="999030 - Disclosure - SELLING AND MARKETING EXPENSES" />
    <link:definitionLink xlink:type="extended" xlink:role="http://n2off.com/role/GeneralAndAdministrativeExpenses" xlink:title="999031 - Disclosure - GENERAL AND ADMINISTRATIVE EXPENSES" />
    <link:definitionLink xlink:type="extended" xlink:role="http://n2off.com/role/FinancingExpensesIncomeNet" xlink:title="999032 - Disclosure - FINANCING EXPENSES (INCOME), NET" />
    <link:definitionLink xlink:type="extended" xlink:role="http://n2off.com/role/IncomeTax" xlink:title="999033 - Disclosure - INCOME TAX" />
    <link:definitionLink xlink:type="extended" xlink:role="http://n2off.com/role/LossPerShare" xlink:title="999034 - Disclosure - LOSS PER SHARE" />
    <link:definitionLink xlink:type="extended" xlink:role="http://n2off.com/role/RelatedParties" xlink:title="999035 - Disclosure - RELATED PARTIES" />
    <link:definitionLink xlink:type="extended" xlink:role="http://n2off.com/role/SegmentReporting" xlink:title="999036 - Disclosure - SEGMENT REPORTING" />
    <link:definitionLink xlink:type="extended" xlink:role="http://n2off.com/role/SubsequentEvents" xlink:title="999037 - Disclosure - SUBSEQUENT EVENTS" />
    <link:definitionLink xlink:type="extended" xlink:role="http://n2off.com/role/SignificantAccountingPoliciesPolicies" xlink:title="999038 - Disclosure - SIGNIFICANT ACCOUNTING POLICIES (Policies)" />
    <link:definitionLink xlink:type="extended" xlink:role="http://n2off.com/role/SignificantAccountingPoliciesTables" xlink:title="999039 - Disclosure - SIGNIFICANT ACCOUNTING POLICIES (Tables)" />
    <link:definitionLink xlink:type="extended" xlink:role="http://n2off.com/role/OtherCurrentAssetsTables" xlink:title="999040 - Disclosure - OTHER CURRENT ASSETS (Tables)" />
    <link:definitionLink xlink:type="extended" xlink:role="http://n2off.com/role/PropertyAndEquipmentNetTables" xlink:title="999041 - Disclosure - PROPERTY AND EQUIPMENT, NET (Tables)" />
    <link:definitionLink xlink:type="extended" xlink:role="http://n2off.com/role/InvestmentInNonconsolidatedAffiliateTables" xlink:title="999042 - Disclosure - INVESTMENT IN NONCONSOLIDATED AFFILIATE (Tables)" />
    <link:definitionLink xlink:type="extended" xlink:role="http://n2off.com/role/OtherLiabilitiesTables" xlink:title="999043 - Disclosure - OTHER LIABILITIES (Tables)" />
    <link:definitionLink xlink:type="extended" xlink:role="http://n2off.com/role/CommitmentAndContingentLiabilitiesTables" xlink:title="999044 - Disclosure - COMMITMENT AND CONTINGENT LIABILITIES (Tables)" />
    <link:definitionLink xlink:type="extended" xlink:role="http://n2off.com/role/LeasesTables" xlink:title="999045 - Disclosure - LEASES (Tables)" />
    <link:definitionLink xlink:type="extended" xlink:role="http://n2off.com/role/StockOptionsTables" xlink:title="999046 - Disclosure - STOCK OPTIONS (Tables)" />
    <link:definitionLink xlink:type="extended" xlink:role="http://n2off.com/role/CostOfSalesTables" xlink:title="999047 - Disclosure - COST OF SALES (Tables)" />
    <link:definitionLink xlink:type="extended" xlink:role="http://n2off.com/role/ResearchAndDevelopmentExpensesTables" xlink:title="999048 - Disclosure - RESEARCH AND DEVELOPMENT EXPENSES (Tables)" />
    <link:definitionLink xlink:type="extended" xlink:role="http://n2off.com/role/SellingAndMarketingExpensesTables" xlink:title="999049 - Disclosure - SELLING AND MARKETING EXPENSES (Tables)" />
    <link:definitionLink xlink:type="extended" xlink:role="http://n2off.com/role/GeneralAndAdministrativeExpensesTables" xlink:title="999050 - Disclosure - GENERAL AND ADMINISTRATIVE EXPENSES (Tables)" />
    <link:definitionLink xlink:type="extended" xlink:role="http://n2off.com/role/FinancingExpensesIncomeNetTables" xlink:title="999051 - Disclosure - FINANCING EXPENSES (INCOME), NET (Tables)" />
    <link:definitionLink xlink:type="extended" xlink:role="http://n2off.com/role/IncomeTaxTables" xlink:title="999052 - Disclosure - INCOME TAX (Tables)" />
    <link:definitionLink xlink:type="extended" xlink:role="http://n2off.com/role/LossPerShareTables" xlink:title="999053 - Disclosure - LOSS PER SHARE (Tables)" />
    <link:definitionLink xlink:type="extended" xlink:role="http://n2off.com/role/RelatedPartiesTables" xlink:title="999054 - Disclosure - RELATED PARTIES (Tables)" />
    <link:definitionLink xlink:type="extended" xlink:role="http://n2off.com/role/SegmentReportingTables" xlink:title="999055 - Disclosure - SEGMENT REPORTING (Tables)" />
    <link:definitionLink xlink:type="extended" xlink:role="http://n2off.com/role/GeneralDetailsNarrative" xlink:title="999056 - Disclosure - GENERAL (Details Narrative)">
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_StatementLineItems" xlink:label="loc_us-gaapStatementLineItems" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_StatementTable" xlink:label="loc_us-gaapStatementTable" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xbrldt:contextElement="segment" xlink:from="loc_us-gaapStatementLineItems" xlink:to="loc_us-gaapStatementTable" xlink:type="arc" order="10" xbrldt:closed="true" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/srt/2024/elts/srt-2024.xsd#srt_ScheduleOfEquityMethodInvestmentEquityMethodInvesteeNameAxis" xlink:label="loc_srtScheduleOfEquityMethodInvestmentEquityMethodInvesteeNameAxis" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_us-gaapStatementTable" xlink:to="loc_srtScheduleOfEquityMethodInvestmentEquityMethodInvesteeNameAxis" xlink:type="arc" order="1" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/srt/2024/elts/srt-2024.xsd#srt_EquityMethodInvesteeNameDomain" xlink:label="loc_srtEquityMethodInvesteeNameDomain" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_srtScheduleOfEquityMethodInvestmentEquityMethodInvesteeNameAxis" xlink:to="loc_srtEquityMethodInvesteeNameDomain" xlink:type="arc" order="20" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/srt/2024/elts/srt-2024.xsd#srt_EquityMethodInvesteeNameDomain" xlink:label="loc_srtEquityMethodInvesteeNameDomain_10" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xbrldt:contextElement="segment" xlink:from="loc_srtScheduleOfEquityMethodInvestmentEquityMethodInvesteeNameAxis" xlink:to="loc_srtEquityMethodInvesteeNameDomain_10" xlink:type="arc" order="30" />
      <link:loc xlink:type="locator" xlink:href="nito-20241231.xsd#NITO_SaveFoodsLtdMember" xlink:label="loc_NITOSaveFoodsLtdMember_0" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_srtEquityMethodInvesteeNameDomain" xlink:to="loc_NITOSaveFoodsLtdMember_0" xlink:type="arc" order="1" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_EquityMethodInvestmentOwnershipPercentage" xlink:label="loc_us-gaapEquityMethodInvestmentOwnershipPercentage_40" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapStatementLineItems" xlink:to="loc_us-gaapEquityMethodInvestmentOwnershipPercentage_40" xlink:type="arc" order="0" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_StockholdersEquityReverseStockSplit" xlink:label="loc_us-gaapStockholdersEquityReverseStockSplit_40" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapStatementLineItems" xlink:to="loc_us-gaapStockholdersEquityReverseStockSplit_40" xlink:type="arc" order="1" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_RetainedEarningsAccumulatedDeficit" xlink:label="loc_us-gaapRetainedEarningsAccumulatedDeficit_40" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapStatementLineItems" xlink:to="loc_us-gaapRetainedEarningsAccumulatedDeficit_40" xlink:type="arc" order="2" />
    </link:definitionLink>
    <link:definitionLink xlink:type="extended" xlink:role="http://n2off.com/role/ScheduleOfPropertyPlantAndEquipmentDepreciationRatesDetails" xlink:title="999057 - Disclosure - SCHEDULE OF PROPERTY, PLANT AND EQUIPMENT DEPRECIATION RATES (Details)">
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_PropertyPlantAndEquipmentLineItems" xlink:label="loc_us-gaapPropertyPlantAndEquipmentLineItems" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_ScheduleOfPropertyPlantAndEquipmentTable" xlink:label="loc_us-gaapScheduleOfPropertyPlantAndEquipmentTable" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xbrldt:contextElement="segment" xlink:from="loc_us-gaapPropertyPlantAndEquipmentLineItems" xlink:to="loc_us-gaapScheduleOfPropertyPlantAndEquipmentTable" xlink:type="arc" order="10" xbrldt:closed="true" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_PropertyPlantAndEquipmentByTypeAxis" xlink:label="loc_us-gaapPropertyPlantAndEquipmentByTypeAxis" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_us-gaapScheduleOfPropertyPlantAndEquipmentTable" xlink:to="loc_us-gaapPropertyPlantAndEquipmentByTypeAxis" xlink:type="arc" order="1" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_PropertyPlantAndEquipmentTypeDomain" xlink:label="loc_us-gaapPropertyPlantAndEquipmentTypeDomain" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_us-gaapPropertyPlantAndEquipmentByTypeAxis" xlink:to="loc_us-gaapPropertyPlantAndEquipmentTypeDomain" xlink:type="arc" order="20" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_PropertyPlantAndEquipmentTypeDomain" xlink:label="loc_us-gaapPropertyPlantAndEquipmentTypeDomain_10" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xbrldt:contextElement="segment" xlink:from="loc_us-gaapPropertyPlantAndEquipmentByTypeAxis" xlink:to="loc_us-gaapPropertyPlantAndEquipmentTypeDomain_10" xlink:type="arc" order="30" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_FurnitureAndFixturesMember" xlink:label="loc_us-gaapFurnitureAndFixturesMember_0" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapPropertyPlantAndEquipmentTypeDomain" xlink:to="loc_us-gaapFurnitureAndFixturesMember_0" xlink:type="arc" order="1" />
      <link:loc xlink:type="locator" xlink:href="nito-20241231.xsd#NITO_MachinesMember" xlink:label="loc_NITOMachinesMember_20" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapPropertyPlantAndEquipmentTypeDomain" xlink:to="loc_NITOMachinesMember_20" xlink:type="arc" order="21" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_ComputerEquipmentMember" xlink:label="loc_us-gaapComputerEquipmentMember_40" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapPropertyPlantAndEquipmentTypeDomain" xlink:to="loc_us-gaapComputerEquipmentMember_40" xlink:type="arc" order="41" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_VehiclesMember" xlink:label="loc_us-gaapVehiclesMember_50" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapPropertyPlantAndEquipmentTypeDomain" xlink:to="loc_us-gaapVehiclesMember_50" xlink:type="arc" order="51" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/srt/2024/elts/srt-2024.xsd#srt_RangeAxis" xlink:label="loc_srtRangeAxis" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_us-gaapScheduleOfPropertyPlantAndEquipmentTable" xlink:to="loc_srtRangeAxis" xlink:type="arc" order="2" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/srt/2024/elts/srt-2024.xsd#srt_RangeMember" xlink:label="loc_srtRangeMember" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_srtRangeAxis" xlink:to="loc_srtRangeMember" xlink:type="arc" order="20" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/srt/2024/elts/srt-2024.xsd#srt_RangeMember" xlink:label="loc_srtRangeMember_10" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xbrldt:contextElement="segment" xlink:from="loc_srtRangeAxis" xlink:to="loc_srtRangeMember_10" xlink:type="arc" order="30" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/srt/2024/elts/srt-2024.xsd#srt_MinimumMember" xlink:label="loc_srtMinimumMember_0" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_srtRangeMember" xlink:to="loc_srtMinimumMember_0" xlink:type="arc" order="2" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/srt/2024/elts/srt-2024.xsd#srt_MaximumMember" xlink:label="loc_srtMaximumMember_10" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_srtRangeMember" xlink:to="loc_srtMaximumMember_10" xlink:type="arc" order="12" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_PropertyPlantAndEquipmentSalvageValuePercentage" xlink:label="loc_us-gaapPropertyPlantAndEquipmentSalvageValuePercentage_70" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapPropertyPlantAndEquipmentLineItems" xlink:to="loc_us-gaapPropertyPlantAndEquipmentSalvageValuePercentage_70" xlink:type="arc" order="0" />
    </link:definitionLink>
    <link:definitionLink xlink:type="extended" xlink:role="http://n2off.com/role/ScheduleOfFairValueAssetsOnRecurringBasisDetails" xlink:title="999058 - Disclosure - SCHEDULE OF FAIR VALUE ASSETS ON RECURRING BASIS (Details)">
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/srt/2024/elts/srt-2024.xsd#srt_PlatformOperatorCryptoAssetLineItems" xlink:label="loc_srtPlatformOperatorCryptoAssetLineItems" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/srt/2024/elts/srt-2024.xsd#srt_PlatformOperatorCryptoAssetTable" xlink:label="loc_srtPlatformOperatorCryptoAssetTable" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xbrldt:contextElement="segment" xlink:from="loc_srtPlatformOperatorCryptoAssetLineItems" xlink:to="loc_srtPlatformOperatorCryptoAssetTable" xlink:type="arc" order="10" xbrldt:closed="true" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_FairValueByFairValueHierarchyLevelAxis" xlink:label="loc_us-gaapFairValueByFairValueHierarchyLevelAxis" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_srtPlatformOperatorCryptoAssetTable" xlink:to="loc_us-gaapFairValueByFairValueHierarchyLevelAxis" xlink:type="arc" order="1" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_FairValueMeasurementsFairValueHierarchyDomain" xlink:label="loc_us-gaapFairValueMeasurementsFairValueHierarchyDomain" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_us-gaapFairValueByFairValueHierarchyLevelAxis" xlink:to="loc_us-gaapFairValueMeasurementsFairValueHierarchyDomain" xlink:type="arc" order="20" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_FairValueMeasurementsFairValueHierarchyDomain" xlink:label="loc_us-gaapFairValueMeasurementsFairValueHierarchyDomain_10" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xbrldt:contextElement="segment" xlink:from="loc_us-gaapFairValueByFairValueHierarchyLevelAxis" xlink:to="loc_us-gaapFairValueMeasurementsFairValueHierarchyDomain_10" xlink:type="arc" order="30" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_FairValueInputsLevel1Member" xlink:label="loc_us-gaapFairValueInputsLevel1Member_0" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapFairValueMeasurementsFairValueHierarchyDomain" xlink:to="loc_us-gaapFairValueInputsLevel1Member_0" xlink:type="arc" order="1" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_FairValueInputsLevel2Member" xlink:label="loc_us-gaapFairValueInputsLevel2Member_10" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapFairValueMeasurementsFairValueHierarchyDomain" xlink:to="loc_us-gaapFairValueInputsLevel2Member_10" xlink:type="arc" order="11" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_FairValueInputsLevel3Member" xlink:label="loc_us-gaapFairValueInputsLevel3Member_20" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapFairValueMeasurementsFairValueHierarchyDomain" xlink:to="loc_us-gaapFairValueInputsLevel3Member_20" xlink:type="arc" order="21" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_FairValueByAssetClassAxis" xlink:label="loc_us-gaapFairValueByAssetClassAxis" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_srtPlatformOperatorCryptoAssetTable" xlink:to="loc_us-gaapFairValueByAssetClassAxis" xlink:type="arc" order="2" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_FairValueAssetsMeasuredOnRecurringBasisUnobservableInputReconciliationByAssetClassDomain" xlink:label="loc_us-gaapFairValueAssetsMeasuredOnRecurringBasisUnobservableInputReconciliationByAssetClassDomain" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_us-gaapFairValueByAssetClassAxis" xlink:to="loc_us-gaapFairValueAssetsMeasuredOnRecurringBasisUnobservableInputReconciliationByAssetClassDomain" xlink:type="arc" order="20" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_FairValueAssetsMeasuredOnRecurringBasisUnobservableInputReconciliationByAssetClassDomain" xlink:label="loc_us-gaapFairValueAssetsMeasuredOnRecurringBasisUnobservableInputReconciliationByAssetClassDomain_10" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xbrldt:contextElement="segment" xlink:from="loc_us-gaapFairValueByAssetClassAxis" xlink:to="loc_us-gaapFairValueAssetsMeasuredOnRecurringBasisUnobservableInputReconciliationByAssetClassDomain_10" xlink:type="arc" order="30" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_InvestmentsMember" xlink:label="loc_us-gaapInvestmentsMember_0" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapFairValueAssetsMeasuredOnRecurringBasisUnobservableInputReconciliationByAssetClassDomain" xlink:to="loc_us-gaapInvestmentsMember_0" xlink:type="arc" order="2" />
      <link:loc xlink:type="locator" xlink:href="nito-20241231.xsd#NITO_InvestmentInSolterraMember" xlink:label="loc_NITOInvestmentInSolterraMember_40" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapFairValueAssetsMeasuredOnRecurringBasisUnobservableInputReconciliationByAssetClassDomain" xlink:to="loc_NITOInvestmentInSolterraMember_40" xlink:type="arc" order="42" />
      <link:loc xlink:type="locator" xlink:href="nito-20241231.xsd#NITO_SolarPvJointVentureProjectMember" xlink:label="loc_NITOSolarPvJointVentureProjectMember_80" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapFairValueAssetsMeasuredOnRecurringBasisUnobservableInputReconciliationByAssetClassDomain" xlink:to="loc_NITOSolarPvJointVentureProjectMember_80" xlink:type="arc" order="82" />
      <link:loc xlink:type="locator" xlink:href="nito-20241231.xsd#NITO_LoanGrantedMitoCareXMember" xlink:label="loc_NITOLoanGrantedMitoCareXMember_120" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapFairValueAssetsMeasuredOnRecurringBasisUnobservableInputReconciliationByAssetClassDomain" xlink:to="loc_NITOLoanGrantedMitoCareXMember_120" xlink:type="arc" order="122" />
      <link:loc xlink:type="locator" xlink:href="nito-20241231.xsd#NITO_ConvertibleLoansToSolterraMember" xlink:label="loc_NITOConvertibleLoansToSolterraMember_160" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapFairValueAssetsMeasuredOnRecurringBasisUnobservableInputReconciliationByAssetClassDomain" xlink:to="loc_NITOConvertibleLoansToSolterraMember_160" xlink:type="arc" order="162" />
      <link:loc xlink:type="locator" xlink:href="nito-20241231.xsd#NITO_ConvertibleLoansToPlantifyMember" xlink:label="loc_NITOConvertibleLoansToPlantifyMember_280" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapFairValueAssetsMeasuredOnRecurringBasisUnobservableInputReconciliationByAssetClassDomain" xlink:to="loc_NITOConvertibleLoansToPlantifyMember_280" xlink:type="arc" order="282" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_AssetsFairValueDisclosure" xlink:label="loc_us-gaapAssetsFairValueDisclosure_370" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_srtPlatformOperatorCryptoAssetLineItems" xlink:to="loc_us-gaapAssetsFairValueDisclosure_370" xlink:type="arc" order="0" />
    </link:definitionLink>
    <link:definitionLink xlink:type="extended" xlink:role="http://n2off.com/role/ScheduleOfChangesInFairValueOfAssetsDetails" xlink:title="999059 - Disclosure - SCHEDULE OF CHANGES IN FAIR VALUE OF ASSETS (Details)">
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/srt/2024/elts/srt-2024.xsd#srt_PlatformOperatorCryptoAssetLineItems" xlink:label="loc_srtPlatformOperatorCryptoAssetLineItems" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/srt/2024/elts/srt-2024.xsd#srt_PlatformOperatorCryptoAssetTable" xlink:label="loc_srtPlatformOperatorCryptoAssetTable" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xbrldt:contextElement="segment" xlink:from="loc_srtPlatformOperatorCryptoAssetLineItems" xlink:to="loc_srtPlatformOperatorCryptoAssetTable" xlink:type="arc" order="10" xbrldt:closed="true" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_FairValueByFairValueHierarchyLevelAxis" xlink:label="loc_us-gaapFairValueByFairValueHierarchyLevelAxis" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_srtPlatformOperatorCryptoAssetTable" xlink:to="loc_us-gaapFairValueByFairValueHierarchyLevelAxis" xlink:type="arc" order="1" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_FairValueMeasurementsFairValueHierarchyDomain" xlink:label="loc_us-gaapFairValueMeasurementsFairValueHierarchyDomain" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_us-gaapFairValueByFairValueHierarchyLevelAxis" xlink:to="loc_us-gaapFairValueMeasurementsFairValueHierarchyDomain" xlink:type="arc" order="20" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_FairValueMeasurementsFairValueHierarchyDomain" xlink:label="loc_us-gaapFairValueMeasurementsFairValueHierarchyDomain_10" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xbrldt:contextElement="segment" xlink:from="loc_us-gaapFairValueByFairValueHierarchyLevelAxis" xlink:to="loc_us-gaapFairValueMeasurementsFairValueHierarchyDomain_10" xlink:type="arc" order="30" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_FairValueInputsLevel1Member" xlink:label="loc_us-gaapFairValueInputsLevel1Member_0" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapFairValueMeasurementsFairValueHierarchyDomain" xlink:to="loc_us-gaapFairValueInputsLevel1Member_0" xlink:type="arc" order="1" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_FairValueInputsLevel3Member" xlink:label="loc_us-gaapFairValueInputsLevel3Member_30" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapFairValueMeasurementsFairValueHierarchyDomain" xlink:to="loc_us-gaapFairValueInputsLevel3Member_30" xlink:type="arc" order="31" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_InvestmentsFairValueDisclosure" xlink:label="loc_us-gaapInvestmentsFairValueDisclosure_70" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_srtPlatformOperatorCryptoAssetLineItems" xlink:to="loc_us-gaapInvestmentsFairValueDisclosure_70" xlink:type="arc" order="0" />
      <link:loc xlink:type="locator" xlink:href="nito-20241231.xsd#NITO_FairValueOfShareIssuedUnderSettlementAgreement" xlink:label="loc_NITOFairValueOfShareIssuedUnderSettlementAgreement_70" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_srtPlatformOperatorCryptoAssetLineItems" xlink:to="loc_NITOFairValueOfShareIssuedUnderSettlementAgreement_70" xlink:type="arc" order="1" />
      <link:loc xlink:type="locator" xlink:href="nito-20241231.xsd#NITO_FairValueMeasurementWithUnobservableInputsReconciliationRecurringBasisInvestmentInSolterra" xlink:label="loc_NITOFairValueMeasurementWithUnobservableInputsReconciliationRecurringBasisInvestmentInSolterra_70" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_srtPlatformOperatorCryptoAssetLineItems" xlink:to="loc_NITOFairValueMeasurementWithUnobservableInputsReconciliationRecurringBasisInvestmentInSolterra_70" xlink:type="arc" order="2" />
      <link:loc xlink:type="locator" xlink:href="nito-20241231.xsd#NITO_GainOnMarketableSecurities" xlink:label="loc_NITOGainOnMarketableSecurities_70" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_srtPlatformOperatorCryptoAssetLineItems" xlink:to="loc_NITOGainOnMarketableSecurities_70" xlink:type="arc" order="3" />
      <link:loc xlink:type="locator" xlink:href="nito-20241231.xsd#NITO_ChangesInFairValueOfAssets" xlink:label="loc_NITOChangesInFairValueOfAssets_70" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_srtPlatformOperatorCryptoAssetLineItems" xlink:to="loc_NITOChangesInFairValueOfAssets_70" xlink:type="arc" order="4" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_FairValueMeasurementWithUnobservableInputsReconciliationRecurringBasisAssetValue" xlink:label="loc_us-gaapFairValueMeasurementWithUnobservableInputsReconciliationRecurringBasisAssetValue_70" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_srtPlatformOperatorCryptoAssetLineItems" xlink:to="loc_us-gaapFairValueMeasurementWithUnobservableInputsReconciliationRecurringBasisAssetValue_70" xlink:type="arc" order="6" />
      <link:loc xlink:type="locator" xlink:href="nito-20241231.xsd#NITO_FairValueMeasurementWithUnobservableInputsReconciliationRecurringBasisCreditFacilityToPlantify" xlink:label="loc_NITOFairValueMeasurementWithUnobservableInputsReconciliationRecurringBasisCreditFacilityToPlantify_70" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_srtPlatformOperatorCryptoAssetLineItems" xlink:to="loc_NITOFairValueMeasurementWithUnobservableInputsReconciliationRecurringBasisCreditFacilityToPlantify_70" xlink:type="arc" order="7" />
      <link:loc xlink:type="locator" xlink:href="nito-20241231.xsd#NITO_FairValueMeasurementWithUnobservableInputsReconciliationRecurringBasisLoansGrantedToSolterra" xlink:label="loc_NITOFairValueMeasurementWithUnobservableInputsReconciliationRecurringBasisLoansGrantedToSolterra_70" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_srtPlatformOperatorCryptoAssetLineItems" xlink:to="loc_NITOFairValueMeasurementWithUnobservableInputsReconciliationRecurringBasisLoansGrantedToSolterra_70" xlink:type="arc" order="8" />
      <link:loc xlink:type="locator" xlink:href="nito-20241231.xsd#NITO_FairValueMeasurementWithUnobservableInputsReconciliationRecurringBasisLoansGrantedToSolarProject" xlink:label="loc_NITOFairValueMeasurementWithUnobservableInputsReconciliationRecurringBasisLoansGrantedToSolarProject_70" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_srtPlatformOperatorCryptoAssetLineItems" xlink:to="loc_NITOFairValueMeasurementWithUnobservableInputsReconciliationRecurringBasisLoansGrantedToSolarProject_70" xlink:type="arc" order="9" />
      <link:loc xlink:type="locator" xlink:href="nito-20241231.xsd#NITO_FairValueMeasurementWithUnobservableInputsReconciliationRecurringBasisLoansGrantedToMitoCareX" xlink:label="loc_NITOFairValueMeasurementWithUnobservableInputsReconciliationRecurringBasisLoansGrantedToMitoCareX_70" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_srtPlatformOperatorCryptoAssetLineItems" xlink:to="loc_NITOFairValueMeasurementWithUnobservableInputsReconciliationRecurringBasisLoansGrantedToMitoCareX_70" xlink:type="arc" order="10" />
      <link:loc xlink:type="locator" xlink:href="nito-20241231.xsd#NITO_FairValueMeasurementWithUnobservableInputsReconciliationRecurringBasisShareIssuedUnderSettlementAgreementInExchangeForInvestment" xlink:label="loc_NITOFairValueMeasurementWithUnobservableInputsReconciliationRecurringBasisShareIssuedUnderSettlementAgreementInExchangeForInvestment_70" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_srtPlatformOperatorCryptoAssetLineItems" xlink:to="loc_NITOFairValueMeasurementWithUnobservableInputsReconciliationRecurringBasisShareIssuedUnderSettlementAgreementInExchangeForInvestment_70" xlink:type="arc" order="11" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_FairValueMeasurementWithUnobservableInputsReconciliationRecurringBasisAssetPeriodIncreaseDecrease" xlink:label="loc_us-gaapFairValueMeasurementWithUnobservableInputsReconciliationRecurringBasisAssetPeriodIncreaseDecrease_70" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_srtPlatformOperatorCryptoAssetLineItems" xlink:to="loc_us-gaapFairValueMeasurementWithUnobservableInputsReconciliationRecurringBasisAssetPeriodIncreaseDecrease_70" xlink:type="arc" order="12" />
    </link:definitionLink>
    <link:definitionLink xlink:type="extended" xlink:role="http://n2off.com/role/SignificantAccountingPoliciesDetailsNarrative" xlink:title="999060 - Disclosure - SIGNIFICANT ACCOUNTING POLICIES (Details Narrative)" />
    <link:definitionLink xlink:type="extended" xlink:role="http://n2off.com/role/ScheduleOfOtherCurrentAssetsDetails" xlink:title="999061 - Disclosure - SCHEDULE OF OTHER CURRENT ASSETS (Details)">
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_DebtSecuritiesHeldtomaturityAllowanceForCreditLossLineItems" xlink:label="loc_us-gaapDebtSecuritiesHeldtomaturityAllowanceForCreditLossLineItems" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_DebtSecuritiesHeldToMaturityAllowanceForCreditLossTable" xlink:label="loc_us-gaapDebtSecuritiesHeldToMaturityAllowanceForCreditLossTable" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xbrldt:contextElement="segment" xlink:from="loc_us-gaapDebtSecuritiesHeldtomaturityAllowanceForCreditLossLineItems" xlink:to="loc_us-gaapDebtSecuritiesHeldToMaturityAllowanceForCreditLossTable" xlink:type="arc" order="10" xbrldt:closed="true" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_FinancialInstrumentAxis" xlink:label="loc_us-gaapFinancialInstrumentAxis" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_us-gaapDebtSecuritiesHeldToMaturityAllowanceForCreditLossTable" xlink:to="loc_us-gaapFinancialInstrumentAxis" xlink:type="arc" order="1" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_TransfersAndServicingOfFinancialInstrumentsTypesOfFinancialInstrumentsDomain" xlink:label="loc_us-gaapTransfersAndServicingOfFinancialInstrumentsTypesOfFinancialInstrumentsDomain" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_us-gaapFinancialInstrumentAxis" xlink:to="loc_us-gaapTransfersAndServicingOfFinancialInstrumentsTypesOfFinancialInstrumentsDomain" xlink:type="arc" order="20" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_TransfersAndServicingOfFinancialInstrumentsTypesOfFinancialInstrumentsDomain" xlink:label="loc_us-gaapTransfersAndServicingOfFinancialInstrumentsTypesOfFinancialInstrumentsDomain_10" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xbrldt:contextElement="segment" xlink:from="loc_us-gaapFinancialInstrumentAxis" xlink:to="loc_us-gaapTransfersAndServicingOfFinancialInstrumentsTypesOfFinancialInstrumentsDomain_10" xlink:type="arc" order="30" />
      <link:loc xlink:type="locator" xlink:href="nito-20241231.xsd#NITO_GovernmentInstitutionsMember" xlink:label="loc_NITOGovernmentInstitutionsMember_0" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapTransfersAndServicingOfFinancialInstrumentsTypesOfFinancialInstrumentsDomain" xlink:to="loc_NITOGovernmentInstitutionsMember_0" xlink:type="arc" order="1" />
      <link:loc xlink:type="locator" xlink:href="nito-20241231.xsd#NITO_OtherReceivableMember" xlink:label="loc_NITOOtherReceivableMember_20" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapTransfersAndServicingOfFinancialInstrumentsTypesOfFinancialInstrumentsDomain" xlink:to="loc_NITOOtherReceivableMember_20" xlink:type="arc" order="21" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_OtherAssetsCurrent" xlink:label="loc_us-gaapOtherAssetsCurrent_70" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapDebtSecuritiesHeldtomaturityAllowanceForCreditLossLineItems" xlink:to="loc_us-gaapOtherAssetsCurrent_70" xlink:type="arc" order="0" />
    </link:definitionLink>
    <link:definitionLink xlink:type="extended" xlink:role="http://n2off.com/role/MitocarexTransactionDetailsNarrative" xlink:title="999062 - Disclosure - MITOCAREX TRANSACTION (Details Narrative)">
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_CollaborativeArrangementsAndNoncollaborativeArrangementTransactionsLineItems" xlink:label="loc_us-gaapCollaborativeArrangementsAndNoncollaborativeArrangementTransactionsLineItems" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_ScheduleOfCollaborativeArrangementsAndNoncollaborativeArrangementTransactionsTable" xlink:label="loc_us-gaapScheduleOfCollaborativeArrangementsAndNoncollaborativeArrangementTransactionsTable" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xbrldt:contextElement="segment" xlink:from="loc_us-gaapCollaborativeArrangementsAndNoncollaborativeArrangementTransactionsLineItems" xlink:to="loc_us-gaapScheduleOfCollaborativeArrangementsAndNoncollaborativeArrangementTransactionsTable" xlink:type="arc" order="10" xbrldt:closed="true" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_TypeOfArrangementAxis" xlink:label="loc_us-gaapTypeOfArrangementAxis" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_us-gaapScheduleOfCollaborativeArrangementsAndNoncollaborativeArrangementTransactionsTable" xlink:to="loc_us-gaapTypeOfArrangementAxis" xlink:type="arc" order="1" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_ArrangementsAndNonarrangementTransactionsMember" xlink:label="loc_us-gaapArrangementsAndNonarrangementTransactionsMember" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_us-gaapTypeOfArrangementAxis" xlink:to="loc_us-gaapArrangementsAndNonarrangementTransactionsMember" xlink:type="arc" order="20" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_ArrangementsAndNonarrangementTransactionsMember" xlink:label="loc_us-gaapArrangementsAndNonarrangementTransactionsMember_10" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xbrldt:contextElement="segment" xlink:from="loc_us-gaapTypeOfArrangementAxis" xlink:to="loc_us-gaapArrangementsAndNonarrangementTransactionsMember_10" xlink:type="arc" order="30" />
      <link:loc xlink:type="locator" xlink:href="nito-20241231.xsd#NITO_LoanAgreementMember" xlink:label="loc_NITOLoanAgreementMember_0" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapArrangementsAndNonarrangementTransactionsMember" xlink:to="loc_NITOLoanAgreementMember_0" xlink:type="arc" order="1" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/srt/2024/elts/srt-2024.xsd#srt_OwnershipAxis" xlink:label="loc_srtOwnershipAxis" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_us-gaapScheduleOfCollaborativeArrangementsAndNoncollaborativeArrangementTransactionsTable" xlink:to="loc_srtOwnershipAxis" xlink:type="arc" order="2" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/srt/2024/elts/srt-2024.xsd#srt_OwnershipDomain" xlink:label="loc_srtOwnershipDomain" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_srtOwnershipAxis" xlink:to="loc_srtOwnershipDomain" xlink:type="arc" order="20" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/srt/2024/elts/srt-2024.xsd#srt_OwnershipDomain" xlink:label="loc_srtOwnershipDomain_10" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xbrldt:contextElement="segment" xlink:from="loc_srtOwnershipAxis" xlink:to="loc_srtOwnershipDomain_10" xlink:type="arc" order="30" />
      <link:loc xlink:type="locator" xlink:href="nito-20241231.xsd#NITO_MitoCareXBioLtdMember" xlink:label="loc_NITOMitoCareXBioLtdMember_0" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_srtOwnershipDomain" xlink:to="loc_NITOMitoCareXBioLtdMember_0" xlink:type="arc" order="2" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_ProceedsFromLoans" xlink:label="loc_us-gaapProceedsFromLoans_50" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapCollaborativeArrangementsAndNoncollaborativeArrangementTransactionsLineItems" xlink:to="loc_us-gaapProceedsFromLoans_50" xlink:type="arc" order="0" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_LongTermDebtPercentageBearingVariableInterestRate" xlink:label="loc_us-gaapLongTermDebtPercentageBearingVariableInterestRate_50" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapCollaborativeArrangementsAndNoncollaborativeArrangementTransactionsLineItems" xlink:to="loc_us-gaapLongTermDebtPercentageBearingVariableInterestRate_50" xlink:type="arc" order="1" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_DebtInstrumentInterestRateEffectivePercentage" xlink:label="loc_us-gaapDebtInstrumentInterestRateEffectivePercentage_50" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapCollaborativeArrangementsAndNoncollaborativeArrangementTransactionsLineItems" xlink:to="loc_us-gaapDebtInstrumentInterestRateEffectivePercentage_50" xlink:type="arc" order="2" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_ShortTermInvestments" xlink:label="loc_us-gaapShortTermInvestments_50" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapCollaborativeArrangementsAndNoncollaborativeArrangementTransactionsLineItems" xlink:to="loc_us-gaapShortTermInvestments_50" xlink:type="arc" order="3" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_InterestExpense" xlink:label="loc_us-gaapInterestExpense_50" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapCollaborativeArrangementsAndNoncollaborativeArrangementTransactionsLineItems" xlink:to="loc_us-gaapInterestExpense_50" xlink:type="arc" order="4" />
    </link:definitionLink>
    <link:definitionLink xlink:type="extended" xlink:role="http://n2off.com/role/ScheduleOfPropertyPlantAndEquipmentDetails" xlink:title="999063 - Disclosure - SCHEDULE OF PROPERTY PLANT AND EQUIPMENT (Details)">
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_PropertyPlantAndEquipmentLineItems" xlink:label="loc_us-gaapPropertyPlantAndEquipmentLineItems" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_ScheduleOfPropertyPlantAndEquipmentTable" xlink:label="loc_us-gaapScheduleOfPropertyPlantAndEquipmentTable" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xbrldt:contextElement="segment" xlink:from="loc_us-gaapPropertyPlantAndEquipmentLineItems" xlink:to="loc_us-gaapScheduleOfPropertyPlantAndEquipmentTable" xlink:type="arc" order="10" xbrldt:closed="true" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_PropertyPlantAndEquipmentByTypeAxis" xlink:label="loc_us-gaapPropertyPlantAndEquipmentByTypeAxis" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_us-gaapScheduleOfPropertyPlantAndEquipmentTable" xlink:to="loc_us-gaapPropertyPlantAndEquipmentByTypeAxis" xlink:type="arc" order="1" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_PropertyPlantAndEquipmentTypeDomain" xlink:label="loc_us-gaapPropertyPlantAndEquipmentTypeDomain" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_us-gaapPropertyPlantAndEquipmentByTypeAxis" xlink:to="loc_us-gaapPropertyPlantAndEquipmentTypeDomain" xlink:type="arc" order="20" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_PropertyPlantAndEquipmentTypeDomain" xlink:label="loc_us-gaapPropertyPlantAndEquipmentTypeDomain_10" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xbrldt:contextElement="segment" xlink:from="loc_us-gaapPropertyPlantAndEquipmentByTypeAxis" xlink:to="loc_us-gaapPropertyPlantAndEquipmentTypeDomain_10" xlink:type="arc" order="30" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_ComputerEquipmentMember" xlink:label="loc_us-gaapComputerEquipmentMember_0" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapPropertyPlantAndEquipmentTypeDomain" xlink:to="loc_us-gaapComputerEquipmentMember_0" xlink:type="arc" order="1" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_FurnitureAndFixturesMember" xlink:label="loc_us-gaapFurnitureAndFixturesMember_20" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapPropertyPlantAndEquipmentTypeDomain" xlink:to="loc_us-gaapFurnitureAndFixturesMember_20" xlink:type="arc" order="21" />
      <link:loc xlink:type="locator" xlink:href="nito-20241231.xsd#NITO_MachinesMember" xlink:label="loc_NITOMachinesMember_40" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapPropertyPlantAndEquipmentTypeDomain" xlink:to="loc_NITOMachinesMember_40" xlink:type="arc" order="41" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_VehiclesMember" xlink:label="loc_us-gaapVehiclesMember_60" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapPropertyPlantAndEquipmentTypeDomain" xlink:to="loc_us-gaapVehiclesMember_60" xlink:type="arc" order="61" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_PropertyPlantAndEquipmentGross" xlink:label="loc_us-gaapPropertyPlantAndEquipmentGross_110" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapPropertyPlantAndEquipmentLineItems" xlink:to="loc_us-gaapPropertyPlantAndEquipmentGross_110" xlink:type="arc" order="0" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_AccumulatedDepreciationDepletionAndAmortizationPropertyPlantAndEquipment" xlink:label="loc_us-gaapAccumulatedDepreciationDepletionAndAmortizationPropertyPlantAndEquipment_110" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapPropertyPlantAndEquipmentLineItems" xlink:to="loc_us-gaapAccumulatedDepreciationDepletionAndAmortizationPropertyPlantAndEquipment_110" xlink:type="arc" order="1" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_PropertyPlantAndEquipmentNet" xlink:label="loc_us-gaapPropertyPlantAndEquipmentNet_110" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapPropertyPlantAndEquipmentLineItems" xlink:to="loc_us-gaapPropertyPlantAndEquipmentNet_110" xlink:type="arc" order="2" />
    </link:definitionLink>
    <link:definitionLink xlink:type="extended" xlink:role="http://n2off.com/role/PropertyAndEquipmentNetDetailsNarrative" xlink:title="999064 - Disclosure - PROPERTY AND EQUIPMENT, NET (Details Narrative)" />
    <link:definitionLink xlink:type="extended" xlink:role="http://n2off.com/role/ScheduleOfFairValueConversionDetails" xlink:title="999065 - Disclosure - SCHEDULE OF FAIR VALUE CONVERSION (Details)">
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_FairValueAssetsAndLiabilitiesMeasuredOnRecurringAndNonrecurringBasisValuationTechniquesLineItems" xlink:label="loc_us-gaapFairValueAssetsAndLiabilitiesMeasuredOnRecurringAndNonrecurringBasisValuationTechniquesLineItems" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_FairValueAssetsAndLiabilitiesMeasuredOnRecurringAndNonrecurringBasisValuationTechniquesTable" xlink:label="loc_us-gaapFairValueAssetsAndLiabilitiesMeasuredOnRecurringAndNonrecurringBasisValuationTechniquesTable" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xbrldt:contextElement="segment" xlink:from="loc_us-gaapFairValueAssetsAndLiabilitiesMeasuredOnRecurringAndNonrecurringBasisValuationTechniquesLineItems" xlink:to="loc_us-gaapFairValueAssetsAndLiabilitiesMeasuredOnRecurringAndNonrecurringBasisValuationTechniquesTable" xlink:type="arc" order="10" xbrldt:closed="true" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_MeasurementInputTypeAxis" xlink:label="loc_us-gaapMeasurementInputTypeAxis" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_us-gaapFairValueAssetsAndLiabilitiesMeasuredOnRecurringAndNonrecurringBasisValuationTechniquesTable" xlink:to="loc_us-gaapMeasurementInputTypeAxis" xlink:type="arc" order="1" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_MeasurementInputTypeDomain" xlink:label="loc_us-gaapMeasurementInputTypeDomain" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_us-gaapMeasurementInputTypeAxis" xlink:to="loc_us-gaapMeasurementInputTypeDomain" xlink:type="arc" order="20" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_MeasurementInputTypeDomain" xlink:label="loc_us-gaapMeasurementInputTypeDomain_10" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xbrldt:contextElement="segment" xlink:from="loc_us-gaapMeasurementInputTypeAxis" xlink:to="loc_us-gaapMeasurementInputTypeDomain_10" xlink:type="arc" order="30" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_MeasurementInputPriceVolatilityMember" xlink:label="loc_us-gaapMeasurementInputPriceVolatilityMember_0" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapMeasurementInputTypeDomain" xlink:to="loc_us-gaapMeasurementInputPriceVolatilityMember_0" xlink:type="arc" order="1" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_MeasurementInputRiskFreeInterestRateMember" xlink:label="loc_us-gaapMeasurementInputRiskFreeInterestRateMember_10" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapMeasurementInputTypeDomain" xlink:to="loc_us-gaapMeasurementInputRiskFreeInterestRateMember_10" xlink:type="arc" order="11" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_MeasurementInputExpectedDividendRateMember" xlink:label="loc_us-gaapMeasurementInputExpectedDividendRateMember_20" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapMeasurementInputTypeDomain" xlink:to="loc_us-gaapMeasurementInputExpectedDividendRateMember_20" xlink:type="arc" order="21" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_MeasurementInputExpectedTermMember" xlink:label="loc_us-gaapMeasurementInputExpectedTermMember_30" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapMeasurementInputTypeDomain" xlink:to="loc_us-gaapMeasurementInputExpectedTermMember_30" xlink:type="arc" order="31" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_MeasurementInputConversionPriceMember" xlink:label="loc_us-gaapMeasurementInputConversionPriceMember_40" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapMeasurementInputTypeDomain" xlink:to="loc_us-gaapMeasurementInputConversionPriceMember_40" xlink:type="arc" order="41" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_MeasurementInputSharePriceMember" xlink:label="loc_us-gaapMeasurementInputSharePriceMember_50" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapMeasurementInputTypeDomain" xlink:to="loc_us-gaapMeasurementInputSharePriceMember_50" xlink:type="arc" order="51" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_EquitySecuritiesFvNiMeasurementInput" xlink:label="loc_us-gaapEquitySecuritiesFvNiMeasurementInput_80" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapFairValueAssetsAndLiabilitiesMeasuredOnRecurringAndNonrecurringBasisValuationTechniquesLineItems" xlink:to="loc_us-gaapEquitySecuritiesFvNiMeasurementInput_80" xlink:type="arc" order="0" />
      <link:loc xlink:type="locator" xlink:href="nito-20241231.xsd#NITO_EquitySecuritiesFvNiMeasurementInputTerm" xlink:label="loc_NITOEquitySecuritiesFvNiMeasurementInputTerm_80" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapFairValueAssetsAndLiabilitiesMeasuredOnRecurringAndNonrecurringBasisValuationTechniquesLineItems" xlink:to="loc_NITOEquitySecuritiesFvNiMeasurementInputTerm_80" xlink:type="arc" order="1" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_EquitySecuritiesFvNiCurrentAndNoncurrent" xlink:label="loc_us-gaapEquitySecuritiesFvNiCurrentAndNoncurrent_80" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapFairValueAssetsAndLiabilitiesMeasuredOnRecurringAndNonrecurringBasisValuationTechniquesLineItems" xlink:to="loc_us-gaapEquitySecuritiesFvNiCurrentAndNoncurrent_80" xlink:type="arc" order="2" />
    </link:definitionLink>
    <link:definitionLink xlink:type="extended" xlink:role="http://n2off.com/role/ScheduleOfEquityInvestmentDetails" xlink:title="999066 - Disclosure - SCHEDULE OF EQUITY INVESTMENT (Details)">
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_StatementLineItems" xlink:label="loc_us-gaapStatementLineItems" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_StatementTable" xlink:label="loc_us-gaapStatementTable" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xbrldt:contextElement="segment" xlink:from="loc_us-gaapStatementLineItems" xlink:to="loc_us-gaapStatementTable" xlink:type="arc" order="10" xbrldt:closed="true" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.sec.gov/dei/2024/dei-2024.xsd#dei_LegalEntityAxis" xlink:label="loc_deiLegalEntityAxis" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_us-gaapStatementTable" xlink:to="loc_deiLegalEntityAxis" xlink:type="arc" order="1" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.sec.gov/dei/2024/dei-2024.xsd#dei_EntityDomain" xlink:label="loc_deiEntityDomain" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_deiLegalEntityAxis" xlink:to="loc_deiEntityDomain" xlink:type="arc" order="20" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.sec.gov/dei/2024/dei-2024.xsd#dei_EntityDomain" xlink:label="loc_deiEntityDomain_10" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xbrldt:contextElement="segment" xlink:from="loc_deiLegalEntityAxis" xlink:to="loc_deiEntityDomain_10" xlink:type="arc" order="30" />
      <link:loc xlink:type="locator" xlink:href="nito-20241231.xsd#NITO_PlantifyFoodsIncMember" xlink:label="loc_NITOPlantifyFoodsIncMember_0" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_deiEntityDomain" xlink:to="loc_NITOPlantifyFoodsIncMember_0" xlink:type="arc" order="1" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_Revenues" xlink:label="loc_us-gaapRevenues_50" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapStatementLineItems" xlink:to="loc_us-gaapRevenues_50" xlink:type="arc" order="0" />
      <link:loc xlink:type="locator" xlink:href="nito-20241231.xsd#NITO_GrossLoss" xlink:label="loc_NITOGrossLoss_50" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapStatementLineItems" xlink:to="loc_NITOGrossLoss_50" xlink:type="arc" order="1" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_NetIncomeLoss" xlink:label="loc_us-gaapNetIncomeLoss_50" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapStatementLineItems" xlink:to="loc_us-gaapNetIncomeLoss_50" xlink:type="arc" order="2" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_AssetsCurrent" xlink:label="loc_us-gaapAssetsCurrent_50" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapStatementLineItems" xlink:to="loc_us-gaapAssetsCurrent_50" xlink:type="arc" order="3" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_AssetsNoncurrent" xlink:label="loc_us-gaapAssetsNoncurrent_50" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapStatementLineItems" xlink:to="loc_us-gaapAssetsNoncurrent_50" xlink:type="arc" order="4" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_LiabilitiesCurrent" xlink:label="loc_us-gaapLiabilitiesCurrent_50" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapStatementLineItems" xlink:to="loc_us-gaapLiabilitiesCurrent_50" xlink:type="arc" order="5" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_StockholdersEquity" xlink:label="loc_us-gaapStockholdersEquity_50" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapStatementLineItems" xlink:to="loc_us-gaapStockholdersEquity_50" xlink:type="arc" order="6" />
    </link:definitionLink>
    <link:definitionLink xlink:type="extended" xlink:role="http://n2off.com/role/InvestmentInNonconsolidatedAffiliateDetailsNarrative" xlink:title="999067 - Disclosure - INVESTMENT IN NONCONSOLIDATED AFFILIATE (Details Narrative)">
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_StatementLineItems" xlink:label="loc_us-gaapStatementLineItems" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_StatementTable" xlink:label="loc_us-gaapStatementTable" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xbrldt:contextElement="segment" xlink:from="loc_us-gaapStatementLineItems" xlink:to="loc_us-gaapStatementTable" xlink:type="arc" order="10" xbrldt:closed="true" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_RelatedPartyTransactionsByRelatedPartyAxis" xlink:label="loc_us-gaapRelatedPartyTransactionsByRelatedPartyAxis" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_us-gaapStatementTable" xlink:to="loc_us-gaapRelatedPartyTransactionsByRelatedPartyAxis" xlink:type="arc" order="1" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_RelatedPartyDomain" xlink:label="loc_us-gaapRelatedPartyDomain" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_us-gaapRelatedPartyTransactionsByRelatedPartyAxis" xlink:to="loc_us-gaapRelatedPartyDomain" xlink:type="arc" order="20" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_RelatedPartyDomain" xlink:label="loc_us-gaapRelatedPartyDomain_10" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xbrldt:contextElement="segment" xlink:from="loc_us-gaapRelatedPartyTransactionsByRelatedPartyAxis" xlink:to="loc_us-gaapRelatedPartyDomain_10" xlink:type="arc" order="30" />
      <link:loc xlink:type="locator" xlink:href="nito-20241231.xsd#NITO_PlantifyFoodsIncMember" xlink:label="loc_NITOPlantifyFoodsIncMember_0" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapRelatedPartyDomain" xlink:to="loc_NITOPlantifyFoodsIncMember_0" xlink:type="arc" order="1" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_StatementEquityComponentsAxis" xlink:label="loc_us-gaapStatementEquityComponentsAxis" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_us-gaapStatementTable" xlink:to="loc_us-gaapStatementEquityComponentsAxis" xlink:type="arc" order="2" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_EquityComponentDomain" xlink:label="loc_us-gaapEquityComponentDomain" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_us-gaapStatementEquityComponentsAxis" xlink:to="loc_us-gaapEquityComponentDomain" xlink:type="arc" order="20" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_EquityComponentDomain" xlink:label="loc_us-gaapEquityComponentDomain_10" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xbrldt:contextElement="segment" xlink:from="loc_us-gaapStatementEquityComponentsAxis" xlink:to="loc_us-gaapEquityComponentDomain_10" xlink:type="arc" order="30" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_CommonStockMember" xlink:label="loc_us-gaapCommonStockMember_10" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapEquityComponentDomain" xlink:to="loc_us-gaapCommonStockMember_10" xlink:type="arc" order="12" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.sec.gov/dei/2024/dei-2024.xsd#dei_LegalEntityAxis" xlink:label="loc_deiLegalEntityAxis" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_us-gaapStatementTable" xlink:to="loc_deiLegalEntityAxis" xlink:type="arc" order="3" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.sec.gov/dei/2024/dei-2024.xsd#dei_EntityDomain" xlink:label="loc_deiEntityDomain" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_deiLegalEntityAxis" xlink:to="loc_deiEntityDomain" xlink:type="arc" order="20" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.sec.gov/dei/2024/dei-2024.xsd#dei_EntityDomain" xlink:label="loc_deiEntityDomain_10" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xbrldt:contextElement="segment" xlink:from="loc_deiLegalEntityAxis" xlink:to="loc_deiEntityDomain_10" xlink:type="arc" order="30" />
      <link:loc xlink:type="locator" xlink:href="nito-20241231.xsd#NITO_PlantifyFoodsIncMember" xlink:label="loc_NITOPlantifyFoodsIncMember_30" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_deiEntityDomain" xlink:to="loc_NITOPlantifyFoodsIncMember_30" xlink:type="arc" order="31" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/srt/2024/elts/srt-2024.xsd#srt_TitleOfIndividualAxis" xlink:label="loc_srtTitleOfIndividualAxis" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_us-gaapStatementTable" xlink:to="loc_srtTitleOfIndividualAxis" xlink:type="arc" order="4" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/srt/2024/elts/srt-2024.xsd#srt_TitleOfIndividualWithRelationshipToEntityDomain" xlink:label="loc_srtTitleOfIndividualWithRelationshipToEntityDomain" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_srtTitleOfIndividualAxis" xlink:to="loc_srtTitleOfIndividualWithRelationshipToEntityDomain" xlink:type="arc" order="20" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/srt/2024/elts/srt-2024.xsd#srt_TitleOfIndividualWithRelationshipToEntityDomain" xlink:label="loc_srtTitleOfIndividualWithRelationshipToEntityDomain_10" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xbrldt:contextElement="segment" xlink:from="loc_srtTitleOfIndividualAxis" xlink:to="loc_srtTitleOfIndividualWithRelationshipToEntityDomain_10" xlink:type="arc" order="30" />
      <link:loc xlink:type="locator" xlink:href="nito-20241231.xsd#NITO_PlantifyFoodsMember" xlink:label="loc_NITOPlantifyFoodsMember_50" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_srtTitleOfIndividualWithRelationshipToEntityDomain" xlink:to="loc_NITOPlantifyFoodsMember_50" xlink:type="arc" order="51" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_TypeOfArrangementAxis" xlink:label="loc_us-gaapTypeOfArrangementAxis" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_us-gaapStatementTable" xlink:to="loc_us-gaapTypeOfArrangementAxis" xlink:type="arc" order="5" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_ArrangementsAndNonarrangementTransactionsMember" xlink:label="loc_us-gaapArrangementsAndNonarrangementTransactionsMember" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_us-gaapTypeOfArrangementAxis" xlink:to="loc_us-gaapArrangementsAndNonarrangementTransactionsMember" xlink:type="arc" order="20" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_ArrangementsAndNonarrangementTransactionsMember" xlink:label="loc_us-gaapArrangementsAndNonarrangementTransactionsMember_10" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xbrldt:contextElement="segment" xlink:from="loc_us-gaapTypeOfArrangementAxis" xlink:to="loc_us-gaapArrangementsAndNonarrangementTransactionsMember_10" xlink:type="arc" order="30" />
      <link:loc xlink:type="locator" xlink:href="nito-20241231.xsd#NITO_SettlementAgreementMember" xlink:label="loc_NITOSettlementAgreementMember_120" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapArrangementsAndNonarrangementTransactionsMember" xlink:to="loc_NITOSettlementAgreementMember_120" xlink:type="arc" order="121" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_SubsequentEventTypeAxis" xlink:label="loc_us-gaapSubsequentEventTypeAxis" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_us-gaapStatementTable" xlink:to="loc_us-gaapSubsequentEventTypeAxis" xlink:type="arc" order="6" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_SubsequentEventTypeDomain" xlink:label="loc_us-gaapSubsequentEventTypeDomain" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_us-gaapSubsequentEventTypeAxis" xlink:to="loc_us-gaapSubsequentEventTypeDomain" xlink:type="arc" order="20" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_SubsequentEventTypeDomain" xlink:label="loc_us-gaapSubsequentEventTypeDomain_10" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xbrldt:contextElement="segment" xlink:from="loc_us-gaapSubsequentEventTypeAxis" xlink:to="loc_us-gaapSubsequentEventTypeDomain_10" xlink:type="arc" order="30" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_SubsequentEventMember" xlink:label="loc_us-gaapSubsequentEventMember_170" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapSubsequentEventTypeDomain" xlink:to="loc_us-gaapSubsequentEventMember_170" xlink:type="arc" order="172" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_SaleOfStockPercentageOfOwnershipBeforeTransaction" xlink:label="loc_us-gaapSaleOfStockPercentageOfOwnershipBeforeTransaction_210" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapStatementLineItems" xlink:to="loc_us-gaapSaleOfStockPercentageOfOwnershipBeforeTransaction_210" xlink:type="arc" order="0" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_SaleOfStockNumberOfSharesIssuedInTransaction" xlink:label="loc_us-gaapSaleOfStockNumberOfSharesIssuedInTransaction_210" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapStatementLineItems" xlink:to="loc_us-gaapSaleOfStockNumberOfSharesIssuedInTransaction_210" xlink:type="arc" order="1" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_SaleOfStockPercentageOfOwnershipAfterTransaction" xlink:label="loc_us-gaapSaleOfStockPercentageOfOwnershipAfterTransaction_210" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapStatementLineItems" xlink:to="loc_us-gaapSaleOfStockPercentageOfOwnershipAfterTransaction_210" xlink:type="arc" order="2" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_DebtInstrumentFaceAmount" xlink:label="loc_us-gaapDebtInstrumentFaceAmount_210" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapStatementLineItems" xlink:to="loc_us-gaapDebtInstrumentFaceAmount_210" xlink:type="arc" order="3" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_DebtInstrumentInterestRateStatedPercentage" xlink:label="loc_us-gaapDebtInstrumentInterestRateStatedPercentage_210" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapStatementLineItems" xlink:to="loc_us-gaapDebtInstrumentInterestRateStatedPercentage_210" xlink:type="arc" order="4" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_DebtInstrumentConvertibleConversionPrice1" xlink:label="loc_us-gaapDebtInstrumentConvertibleConversionPrice1_210" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapStatementLineItems" xlink:to="loc_us-gaapDebtInstrumentConvertibleConversionPrice1_210" xlink:type="arc" order="5" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_SharePrice" xlink:label="loc_us-gaapSharePrice_210" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapStatementLineItems" xlink:to="loc_us-gaapSharePrice_210" xlink:type="arc" order="6" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_SaleOfStockPricePerShare" xlink:label="loc_us-gaapSaleOfStockPricePerShare_210" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapStatementLineItems" xlink:to="loc_us-gaapSaleOfStockPricePerShare_210" xlink:type="arc" order="7" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_SaleOfStockConsiderationReceivedOnTransaction" xlink:label="loc_us-gaapSaleOfStockConsiderationReceivedOnTransaction_210" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapStatementLineItems" xlink:to="loc_us-gaapSaleOfStockConsiderationReceivedOnTransaction_210" xlink:type="arc" order="8" />
      <link:loc xlink:type="locator" xlink:href="nito-20241231.xsd#NITO_IncreaseInPercentageOfOwnership" xlink:label="loc_NITOIncreaseInPercentageOfOwnership_210" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapStatementLineItems" xlink:to="loc_NITOIncreaseInPercentageOfOwnership_210" xlink:type="arc" order="9" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_InvestmentOwnedBalanceShares" xlink:label="loc_us-gaapInvestmentOwnedBalanceShares_210" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapStatementLineItems" xlink:to="loc_us-gaapInvestmentOwnedBalanceShares_210" xlink:type="arc" order="10" />
      <link:loc xlink:type="locator" xlink:href="nito-20241231.xsd#NITO_DebtInstrumentInvestmentRate" xlink:label="loc_NITODebtInstrumentInvestmentRate_210" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapStatementLineItems" xlink:to="loc_NITODebtInstrumentInvestmentRate_210" xlink:type="arc" order="11" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_LineOfCreditFacilityMaximumBorrowingCapacity" xlink:label="loc_us-gaapLineOfCreditFacilityMaximumBorrowingCapacity_210" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapStatementLineItems" xlink:to="loc_us-gaapLineOfCreditFacilityMaximumBorrowingCapacity_210" xlink:type="arc" order="12" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_LineOfCreditFacilityInterestRateDuringPeriod" xlink:label="loc_us-gaapLineOfCreditFacilityInterestRateDuringPeriod_210" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapStatementLineItems" xlink:to="loc_us-gaapLineOfCreditFacilityInterestRateDuringPeriod_210" xlink:type="arc" order="13" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_StockIssuedDuringPeriodSharesNewIssues" xlink:label="loc_us-gaapStockIssuedDuringPeriodSharesNewIssues_210" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapStatementLineItems" xlink:to="loc_us-gaapStockIssuedDuringPeriodSharesNewIssues_210" xlink:type="arc" order="14" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_DebtInstrumentPeriodicPayment" xlink:label="loc_us-gaapDebtInstrumentPeriodicPayment_210" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapStatementLineItems" xlink:to="loc_us-gaapDebtInstrumentPeriodicPayment_210" xlink:type="arc" order="15" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_DebtInstrumentMaturityDate" xlink:label="loc_us-gaapDebtInstrumentMaturityDate_210" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapStatementLineItems" xlink:to="loc_us-gaapDebtInstrumentMaturityDate_210" xlink:type="arc" order="16" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_DebtInstrumentInterestRateIncreaseDecrease" xlink:label="loc_us-gaapDebtInstrumentInterestRateIncreaseDecrease_210" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapStatementLineItems" xlink:to="loc_us-gaapDebtInstrumentInterestRateIncreaseDecrease_210" xlink:type="arc" order="17" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_TreasuryStockValueAcquiredCostMethod" xlink:label="loc_us-gaapTreasuryStockValueAcquiredCostMethod_210" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapStatementLineItems" xlink:to="loc_us-gaapTreasuryStockValueAcquiredCostMethod_210" xlink:type="arc" order="18" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_UnrealizedGainLossOnInvestments" xlink:label="loc_us-gaapUnrealizedGainLossOnInvestments_210" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapStatementLineItems" xlink:to="loc_us-gaapUnrealizedGainLossOnInvestments_210" xlink:type="arc" order="19" />
    </link:definitionLink>
    <link:definitionLink xlink:type="extended" xlink:role="http://n2off.com/role/InvestmentAndLoanToSolterraDetailsNarrative" xlink:title="999068 - Disclosure - INVESTMENT AND LOAN TO SOLTERRA (Details Narrative)">
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_DebtInstrumentLineItems" xlink:label="loc_us-gaapDebtInstrumentLineItems" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_DebtInstrumentTable" xlink:label="loc_us-gaapDebtInstrumentTable" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xbrldt:contextElement="segment" xlink:from="loc_us-gaapDebtInstrumentLineItems" xlink:to="loc_us-gaapDebtInstrumentTable" xlink:type="arc" order="10" xbrldt:closed="true" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_TypeOfArrangementAxis" xlink:label="loc_us-gaapTypeOfArrangementAxis" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_us-gaapDebtInstrumentTable" xlink:to="loc_us-gaapTypeOfArrangementAxis" xlink:type="arc" order="1" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_ArrangementsAndNonarrangementTransactionsMember" xlink:label="loc_us-gaapArrangementsAndNonarrangementTransactionsMember" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_us-gaapTypeOfArrangementAxis" xlink:to="loc_us-gaapArrangementsAndNonarrangementTransactionsMember" xlink:type="arc" order="20" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_ArrangementsAndNonarrangementTransactionsMember" xlink:label="loc_us-gaapArrangementsAndNonarrangementTransactionsMember_10" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xbrldt:contextElement="segment" xlink:from="loc_us-gaapTypeOfArrangementAxis" xlink:to="loc_us-gaapArrangementsAndNonarrangementTransactionsMember_10" xlink:type="arc" order="30" />
      <link:loc xlink:type="locator" xlink:href="nito-20241231.xsd#NITO_LoanAgreementMember" xlink:label="loc_NITOLoanAgreementMember_0" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapArrangementsAndNonarrangementTransactionsMember" xlink:to="loc_NITOLoanAgreementMember_0" xlink:type="arc" order="1" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_RelatedPartyTransactionsByRelatedPartyAxis" xlink:label="loc_us-gaapRelatedPartyTransactionsByRelatedPartyAxis" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_us-gaapDebtInstrumentTable" xlink:to="loc_us-gaapRelatedPartyTransactionsByRelatedPartyAxis" xlink:type="arc" order="2" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_RelatedPartyDomain" xlink:label="loc_us-gaapRelatedPartyDomain" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_us-gaapRelatedPartyTransactionsByRelatedPartyAxis" xlink:to="loc_us-gaapRelatedPartyDomain" xlink:type="arc" order="20" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_RelatedPartyDomain" xlink:label="loc_us-gaapRelatedPartyDomain_10" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xbrldt:contextElement="segment" xlink:from="loc_us-gaapRelatedPartyTransactionsByRelatedPartyAxis" xlink:to="loc_us-gaapRelatedPartyDomain_10" xlink:type="arc" order="30" />
      <link:loc xlink:type="locator" xlink:href="nito-20241231.xsd#NITO_SolterraRenewableEnergyLtdMember" xlink:label="loc_NITOSolterraRenewableEnergyLtdMember_0" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapRelatedPartyDomain" xlink:to="loc_NITOSolterraRenewableEnergyLtdMember_0" xlink:type="arc" order="2" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/srt/2024/elts/srt-2024.xsd#srt_RangeAxis" xlink:label="loc_srtRangeAxis" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_us-gaapDebtInstrumentTable" xlink:to="loc_srtRangeAxis" xlink:type="arc" order="3" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/srt/2024/elts/srt-2024.xsd#srt_RangeMember" xlink:label="loc_srtRangeMember" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_srtRangeAxis" xlink:to="loc_srtRangeMember" xlink:type="arc" order="20" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/srt/2024/elts/srt-2024.xsd#srt_RangeMember" xlink:label="loc_srtRangeMember_10" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xbrldt:contextElement="segment" xlink:from="loc_srtRangeAxis" xlink:to="loc_srtRangeMember_10" xlink:type="arc" order="30" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/srt/2024/elts/srt-2024.xsd#srt_MinimumMember" xlink:label="loc_srtMinimumMember_20" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_srtRangeMember" xlink:to="loc_srtMinimumMember_20" xlink:type="arc" order="23" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/srt/2024/elts/srt-2024.xsd#srt_MaximumMember" xlink:label="loc_srtMaximumMember_30" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_srtRangeMember" xlink:to="loc_srtMaximumMember_30" xlink:type="arc" order="33" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_BusinessAcquisitionAxis" xlink:label="loc_us-gaapBusinessAcquisitionAxis" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_us-gaapDebtInstrumentTable" xlink:to="loc_us-gaapBusinessAcquisitionAxis" xlink:type="arc" order="4" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_BusinessAcquisitionAcquireeDomain" xlink:label="loc_us-gaapBusinessAcquisitionAcquireeDomain" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_us-gaapBusinessAcquisitionAxis" xlink:to="loc_us-gaapBusinessAcquisitionAcquireeDomain" xlink:type="arc" order="20" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_BusinessAcquisitionAcquireeDomain" xlink:label="loc_us-gaapBusinessAcquisitionAcquireeDomain_10" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xbrldt:contextElement="segment" xlink:from="loc_us-gaapBusinessAcquisitionAxis" xlink:to="loc_us-gaapBusinessAcquisitionAcquireeDomain_10" xlink:type="arc" order="30" />
      <link:loc xlink:type="locator" xlink:href="nito-20241231.xsd#NITO_SolterraEnergyLtdMember" xlink:label="loc_NITOSolterraEnergyLtdMember_70" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapBusinessAcquisitionAcquireeDomain" xlink:to="loc_NITOSolterraEnergyLtdMember_70" xlink:type="arc" order="71" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_DebtInstrumentFaceAmount" xlink:label="loc_us-gaapDebtInstrumentFaceAmount_110" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapDebtInstrumentLineItems" xlink:to="loc_us-gaapDebtInstrumentFaceAmount_110" xlink:type="arc" order="0" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_DebtInstrumentCarryingAmount" xlink:label="loc_us-gaapDebtInstrumentCarryingAmount_110" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapDebtInstrumentLineItems" xlink:to="loc_us-gaapDebtInstrumentCarryingAmount_110" xlink:type="arc" order="1" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_DebtInstrumentInterestRateDuringPeriod" xlink:label="loc_us-gaapDebtInstrumentInterestRateDuringPeriod_110" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapDebtInstrumentLineItems" xlink:to="loc_us-gaapDebtInstrumentInterestRateDuringPeriod_110" xlink:type="arc" order="2" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_RepaymentsOfDebt" xlink:label="loc_us-gaapRepaymentsOfDebt_110" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapDebtInstrumentLineItems" xlink:to="loc_us-gaapRepaymentsOfDebt_110" xlink:type="arc" order="3" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_BusinessAcquisitionEquityInterestsIssuedOrIssuableNumberOfSharesIssued" xlink:label="loc_us-gaapBusinessAcquisitionEquityInterestsIssuedOrIssuableNumberOfSharesIssued_110" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapDebtInstrumentLineItems" xlink:to="loc_us-gaapBusinessAcquisitionEquityInterestsIssuedOrIssuableNumberOfSharesIssued_110" xlink:type="arc" order="4" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_BusinessCombinationConsiderationTransferred1" xlink:label="loc_us-gaapBusinessCombinationConsiderationTransferred1_110" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapDebtInstrumentLineItems" xlink:to="loc_us-gaapBusinessCombinationConsiderationTransferred1_110" xlink:type="arc" order="5" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_ChangeInUnrealizedGainLossOnForeignCurrencyFairValueHedgingInstruments1" xlink:label="loc_us-gaapChangeInUnrealizedGainLossOnForeignCurrencyFairValueHedgingInstruments1_110" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapDebtInstrumentLineItems" xlink:to="loc_us-gaapChangeInUnrealizedGainLossOnForeignCurrencyFairValueHedgingInstruments1_110" xlink:type="arc" order="6" />
    </link:definitionLink>
    <link:definitionLink xlink:type="extended" xlink:role="http://n2off.com/role/SolarPhotovolaicJointVentureProjectDetailsNarrative" xlink:title="999069 - Disclosure - SOLAR PHOTOVOLAIC JOINT VENTURE PROJECT (Details Narrative)">
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_CollaborativeArrangementsAndNoncollaborativeArrangementTransactionsLineItems" xlink:label="loc_us-gaapCollaborativeArrangementsAndNoncollaborativeArrangementTransactionsLineItems" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_ScheduleOfCollaborativeArrangementsAndNoncollaborativeArrangementTransactionsTable" xlink:label="loc_us-gaapScheduleOfCollaborativeArrangementsAndNoncollaborativeArrangementTransactionsTable" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xbrldt:contextElement="segment" xlink:from="loc_us-gaapCollaborativeArrangementsAndNoncollaborativeArrangementTransactionsLineItems" xlink:to="loc_us-gaapScheduleOfCollaborativeArrangementsAndNoncollaborativeArrangementTransactionsTable" xlink:type="arc" order="10" xbrldt:closed="true" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_TypeOfArrangementAxis" xlink:label="loc_us-gaapTypeOfArrangementAxis" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_us-gaapScheduleOfCollaborativeArrangementsAndNoncollaborativeArrangementTransactionsTable" xlink:to="loc_us-gaapTypeOfArrangementAxis" xlink:type="arc" order="1" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_ArrangementsAndNonarrangementTransactionsMember" xlink:label="loc_us-gaapArrangementsAndNonarrangementTransactionsMember" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_us-gaapTypeOfArrangementAxis" xlink:to="loc_us-gaapArrangementsAndNonarrangementTransactionsMember" xlink:type="arc" order="20" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_ArrangementsAndNonarrangementTransactionsMember" xlink:label="loc_us-gaapArrangementsAndNonarrangementTransactionsMember_10" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xbrldt:contextElement="segment" xlink:from="loc_us-gaapTypeOfArrangementAxis" xlink:to="loc_us-gaapArrangementsAndNonarrangementTransactionsMember_10" xlink:type="arc" order="30" />
      <link:loc xlink:type="locator" xlink:href="nito-20241231.xsd#NITO_LoanAndPartnershipAgreementMember" xlink:label="loc_NITOLoanAndPartnershipAgreementMember_0" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapArrangementsAndNonarrangementTransactionsMember" xlink:to="loc_NITOLoanAndPartnershipAgreementMember_0" xlink:type="arc" order="1" />
      <link:loc xlink:type="locator" xlink:href="nito-20241231.xsd#NITO_LoanAgreementMember" xlink:label="loc_NITOLoanAgreementMember_60" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapArrangementsAndNonarrangementTransactionsMember" xlink:to="loc_NITOLoanAgreementMember_60" xlink:type="arc" order="61" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_DebtInstrumentFaceAmount" xlink:label="loc_us-gaapDebtInstrumentFaceAmount_100" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapCollaborativeArrangementsAndNoncollaborativeArrangementTransactionsLineItems" xlink:to="loc_us-gaapDebtInstrumentFaceAmount_100" xlink:type="arc" order="0" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_DebtInstrumentCarryingAmount" xlink:label="loc_us-gaapDebtInstrumentCarryingAmount_100" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapCollaborativeArrangementsAndNoncollaborativeArrangementTransactionsLineItems" xlink:to="loc_us-gaapDebtInstrumentCarryingAmount_100" xlink:type="arc" order="1" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_DebtInstrumentInterestRateStatedPercentage" xlink:label="loc_us-gaapDebtInstrumentInterestRateStatedPercentage_100" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapCollaborativeArrangementsAndNoncollaborativeArrangementTransactionsLineItems" xlink:to="loc_us-gaapDebtInstrumentInterestRateStatedPercentage_100" xlink:type="arc" order="2" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_DebtInstrumentDescription" xlink:label="loc_us-gaapDebtInstrumentDescription_100" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapCollaborativeArrangementsAndNoncollaborativeArrangementTransactionsLineItems" xlink:to="loc_us-gaapDebtInstrumentDescription_100" xlink:type="arc" order="3" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_RepaymentsOfDebt" xlink:label="loc_us-gaapRepaymentsOfDebt_100" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapCollaborativeArrangementsAndNoncollaborativeArrangementTransactionsLineItems" xlink:to="loc_us-gaapRepaymentsOfDebt_100" xlink:type="arc" order="4" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_InterestIncomeOther" xlink:label="loc_us-gaapInterestIncomeOther_100" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapCollaborativeArrangementsAndNoncollaborativeArrangementTransactionsLineItems" xlink:to="loc_us-gaapInterestIncomeOther_100" xlink:type="arc" order="5" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_UnrealizedGainLossOnInvestments" xlink:label="loc_us-gaapUnrealizedGainLossOnInvestments_100" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapCollaborativeArrangementsAndNoncollaborativeArrangementTransactionsLineItems" xlink:to="loc_us-gaapUnrealizedGainLossOnInvestments_100" xlink:type="arc" order="6" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_LoansPayable" xlink:label="loc_us-gaapLoansPayable_100" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapCollaborativeArrangementsAndNoncollaborativeArrangementTransactionsLineItems" xlink:to="loc_us-gaapLoansPayable_100" xlink:type="arc" order="7" />
    </link:definitionLink>
    <link:definitionLink xlink:type="extended" xlink:role="http://n2off.com/role/ConsolidatedEntityDetailsNarrative" xlink:title="999070 - Disclosure - A CONSOLIDATED ENTITY (Details Narrative)">
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_StatementLineItems" xlink:label="loc_us-gaapStatementLineItems" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_StatementTable" xlink:label="loc_us-gaapStatementTable" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xbrldt:contextElement="segment" xlink:from="loc_us-gaapStatementLineItems" xlink:to="loc_us-gaapStatementTable" xlink:type="arc" order="10" xbrldt:closed="true" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/srt/2024/elts/srt-2024.xsd#srt_ScheduleOfEquityMethodInvestmentEquityMethodInvesteeNameAxis" xlink:label="loc_srtScheduleOfEquityMethodInvestmentEquityMethodInvesteeNameAxis" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_us-gaapStatementTable" xlink:to="loc_srtScheduleOfEquityMethodInvestmentEquityMethodInvesteeNameAxis" xlink:type="arc" order="1" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/srt/2024/elts/srt-2024.xsd#srt_EquityMethodInvesteeNameDomain" xlink:label="loc_srtEquityMethodInvesteeNameDomain" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_srtScheduleOfEquityMethodInvestmentEquityMethodInvesteeNameAxis" xlink:to="loc_srtEquityMethodInvesteeNameDomain" xlink:type="arc" order="20" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/srt/2024/elts/srt-2024.xsd#srt_EquityMethodInvesteeNameDomain" xlink:label="loc_srtEquityMethodInvesteeNameDomain_10" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xbrldt:contextElement="segment" xlink:from="loc_srtScheduleOfEquityMethodInvestmentEquityMethodInvesteeNameAxis" xlink:to="loc_srtEquityMethodInvesteeNameDomain_10" xlink:type="arc" order="30" />
      <link:loc xlink:type="locator" xlink:href="nito-20241231.xsd#NITO_YaaranInvestmentMember" xlink:label="loc_NITOYaaranInvestmentMember_0" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_srtEquityMethodInvesteeNameDomain" xlink:to="loc_NITOYaaranInvestmentMember_0" xlink:type="arc" order="1" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/srt/2024/elts/srt-2024.xsd#srt_OwnershipAxis" xlink:label="loc_srtOwnershipAxis" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_us-gaapStatementTable" xlink:to="loc_srtOwnershipAxis" xlink:type="arc" order="2" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/srt/2024/elts/srt-2024.xsd#srt_OwnershipDomain" xlink:label="loc_srtOwnershipDomain" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_srtOwnershipAxis" xlink:to="loc_srtOwnershipDomain" xlink:type="arc" order="20" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/srt/2024/elts/srt-2024.xsd#srt_OwnershipDomain" xlink:label="loc_srtOwnershipDomain_10" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xbrldt:contextElement="segment" xlink:from="loc_srtOwnershipAxis" xlink:to="loc_srtOwnershipDomain_10" xlink:type="arc" order="30" />
      <link:loc xlink:type="locator" xlink:href="nito-20241231.xsd#NITO_NitrousinkLtdMember" xlink:label="loc_NITONitrousinkLtdMember_30" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_srtOwnershipDomain" xlink:to="loc_NITONitrousinkLtdMember_30" xlink:type="arc" order="32" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/srt/2024/elts/srt-2024.xsd#srt_RangeAxis" xlink:label="loc_srtRangeAxis" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_us-gaapStatementTable" xlink:to="loc_srtRangeAxis" xlink:type="arc" order="3" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/srt/2024/elts/srt-2024.xsd#srt_RangeMember" xlink:label="loc_srtRangeMember" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_srtRangeAxis" xlink:to="loc_srtRangeMember" xlink:type="arc" order="20" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/srt/2024/elts/srt-2024.xsd#srt_RangeMember" xlink:label="loc_srtRangeMember_10" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xbrldt:contextElement="segment" xlink:from="loc_srtRangeAxis" xlink:to="loc_srtRangeMember_10" xlink:type="arc" order="30" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/srt/2024/elts/srt-2024.xsd#srt_MaximumMember" xlink:label="loc_srtMaximumMember_50" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_srtRangeMember" xlink:to="loc_srtMaximumMember_50" xlink:type="arc" order="51" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_BusinessAcquisitionAxis" xlink:label="loc_us-gaapBusinessAcquisitionAxis" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_us-gaapStatementTable" xlink:to="loc_us-gaapBusinessAcquisitionAxis" xlink:type="arc" order="4" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_BusinessAcquisitionAcquireeDomain" xlink:label="loc_us-gaapBusinessAcquisitionAcquireeDomain" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_us-gaapBusinessAcquisitionAxis" xlink:to="loc_us-gaapBusinessAcquisitionAcquireeDomain" xlink:type="arc" order="20" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_BusinessAcquisitionAcquireeDomain" xlink:label="loc_us-gaapBusinessAcquisitionAcquireeDomain_10" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xbrldt:contextElement="segment" xlink:from="loc_us-gaapBusinessAcquisitionAxis" xlink:to="loc_us-gaapBusinessAcquisitionAcquireeDomain_10" xlink:type="arc" order="30" />
      <link:loc xlink:type="locator" xlink:href="nito-20241231.xsd#NITO_IPRAndDMember" xlink:label="loc_NITOIPRAndDMember_80" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapBusinessAcquisitionAcquireeDomain" xlink:to="loc_NITOIPRAndDMember_80" xlink:type="arc" order="81" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_SharesIssued" xlink:label="loc_us-gaapSharesIssued_100" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapStatementLineItems" xlink:to="loc_us-gaapSharesIssued_100" xlink:type="arc" order="0" />
      <link:loc xlink:type="locator" xlink:href="nito-20241231.xsd#NITO_ShareCapitalPercentage" xlink:label="loc_NITOShareCapitalPercentage_100" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapStatementLineItems" xlink:to="loc_NITOShareCapitalPercentage_100" xlink:type="arc" order="1" />
      <link:loc xlink:type="locator" xlink:href="nito-20241231.xsd#NITO_ExchangeCapitalForSharePrice" xlink:label="loc_NITOExchangeCapitalForSharePrice_100" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapStatementLineItems" xlink:to="loc_NITOExchangeCapitalForSharePrice_100" xlink:type="arc" order="2" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_EquityMethodInvestmentOwnershipPercentage" xlink:label="loc_us-gaapEquityMethodInvestmentOwnershipPercentage_100" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapStatementLineItems" xlink:to="loc_us-gaapEquityMethodInvestmentOwnershipPercentage_100" xlink:type="arc" order="3" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_MinorityInterestOwnershipPercentageByParent" xlink:label="loc_us-gaapMinorityInterestOwnershipPercentageByParent_100" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapStatementLineItems" xlink:to="loc_us-gaapMinorityInterestOwnershipPercentageByParent_100" xlink:type="arc" order="4" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_NatureOfCommonOwnershipOrManagementControlRelationships" xlink:label="loc_us-gaapNatureOfCommonOwnershipOrManagementControlRelationships_100" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapStatementLineItems" xlink:to="loc_us-gaapNatureOfCommonOwnershipOrManagementControlRelationships_100" xlink:type="arc" order="5" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_ResearchAndDevelopmentExpense" xlink:label="loc_us-gaapResearchAndDevelopmentExpense_100" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapStatementLineItems" xlink:to="loc_us-gaapResearchAndDevelopmentExpense_100" xlink:type="arc" order="6" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_DebtInstrumentPeriodicPayment" xlink:label="loc_us-gaapDebtInstrumentPeriodicPayment_100" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapStatementLineItems" xlink:to="loc_us-gaapDebtInstrumentPeriodicPayment_100" xlink:type="arc" order="7" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_NoncontrollingInterestIncreaseFromSubsidiaryEquityIssuance" xlink:label="loc_us-gaapNoncontrollingInterestIncreaseFromSubsidiaryEquityIssuance_100" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapStatementLineItems" xlink:to="loc_us-gaapNoncontrollingInterestIncreaseFromSubsidiaryEquityIssuance_100" xlink:type="arc" order="8" />
    </link:definitionLink>
    <link:definitionLink xlink:type="extended" xlink:role="http://n2off.com/role/ScheduleOfOtherLiabilitiesDetails" xlink:title="999071 - Disclosure - SCHEDULE OF OTHER LIABILITIES (Details)">
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_DebtSecuritiesHeldtomaturityAllowanceForCreditLossLineItems" xlink:label="loc_us-gaapDebtSecuritiesHeldtomaturityAllowanceForCreditLossLineItems" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_DebtSecuritiesHeldToMaturityAllowanceForCreditLossTable" xlink:label="loc_us-gaapDebtSecuritiesHeldToMaturityAllowanceForCreditLossTable" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xbrldt:contextElement="segment" xlink:from="loc_us-gaapDebtSecuritiesHeldtomaturityAllowanceForCreditLossLineItems" xlink:to="loc_us-gaapDebtSecuritiesHeldToMaturityAllowanceForCreditLossTable" xlink:type="arc" order="10" xbrldt:closed="true" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_FinancialInstrumentAxis" xlink:label="loc_us-gaapFinancialInstrumentAxis" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_us-gaapDebtSecuritiesHeldToMaturityAllowanceForCreditLossTable" xlink:to="loc_us-gaapFinancialInstrumentAxis" xlink:type="arc" order="1" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_TransfersAndServicingOfFinancialInstrumentsTypesOfFinancialInstrumentsDomain" xlink:label="loc_us-gaapTransfersAndServicingOfFinancialInstrumentsTypesOfFinancialInstrumentsDomain" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_us-gaapFinancialInstrumentAxis" xlink:to="loc_us-gaapTransfersAndServicingOfFinancialInstrumentsTypesOfFinancialInstrumentsDomain" xlink:type="arc" order="20" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_TransfersAndServicingOfFinancialInstrumentsTypesOfFinancialInstrumentsDomain" xlink:label="loc_us-gaapTransfersAndServicingOfFinancialInstrumentsTypesOfFinancialInstrumentsDomain_10" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xbrldt:contextElement="segment" xlink:from="loc_us-gaapFinancialInstrumentAxis" xlink:to="loc_us-gaapTransfersAndServicingOfFinancialInstrumentsTypesOfFinancialInstrumentsDomain_10" xlink:type="arc" order="30" />
      <link:loc xlink:type="locator" xlink:href="nito-20241231.xsd#NITO_EmployeesAndRelatedInstitutionsMember" xlink:label="loc_NITOEmployeesAndRelatedInstitutionsMember_0" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapTransfersAndServicingOfFinancialInstrumentsTypesOfFinancialInstrumentsDomain" xlink:to="loc_NITOEmployeesAndRelatedInstitutionsMember_0" xlink:type="arc" order="1" />
      <link:loc xlink:type="locator" xlink:href="nito-20241231.xsd#NITO_AccruedExpensesMember" xlink:label="loc_NITOAccruedExpensesMember_20" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapTransfersAndServicingOfFinancialInstrumentsTypesOfFinancialInstrumentsDomain" xlink:to="loc_NITOAccruedExpensesMember_20" xlink:type="arc" order="21" />
      <link:loc xlink:type="locator" xlink:href="nito-20241231.xsd#NITO_OperatingLeaseLiabilitiesMember" xlink:label="loc_NITOOperatingLeaseLiabilitiesMember_40" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapTransfersAndServicingOfFinancialInstrumentsTypesOfFinancialInstrumentsDomain" xlink:to="loc_NITOOperatingLeaseLiabilitiesMember_40" xlink:type="arc" order="41" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_OtherLiabilitiesCurrent" xlink:label="loc_us-gaapOtherLiabilitiesCurrent_90" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapDebtSecuritiesHeldtomaturityAllowanceForCreditLossLineItems" xlink:to="loc_us-gaapOtherLiabilitiesCurrent_90" xlink:type="arc" order="0" />
    </link:definitionLink>
    <link:definitionLink xlink:type="extended" xlink:role="http://n2off.com/role/ScheduleOfWarrantAssumptionsUsedDetails" xlink:title="999072 - Disclosure - SCHEDULE OF WARRANT ASSUMPTIONS USED (Details)">
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_FairValueAssetsAndLiabilitiesMeasuredOnRecurringAndNonrecurringBasisValuationTechniquesLineItems" xlink:label="loc_us-gaapFairValueAssetsAndLiabilitiesMeasuredOnRecurringAndNonrecurringBasisValuationTechniquesLineItems" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_FairValueAssetsAndLiabilitiesMeasuredOnRecurringAndNonrecurringBasisValuationTechniquesTable" xlink:label="loc_us-gaapFairValueAssetsAndLiabilitiesMeasuredOnRecurringAndNonrecurringBasisValuationTechniquesTable" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xbrldt:contextElement="segment" xlink:from="loc_us-gaapFairValueAssetsAndLiabilitiesMeasuredOnRecurringAndNonrecurringBasisValuationTechniquesLineItems" xlink:to="loc_us-gaapFairValueAssetsAndLiabilitiesMeasuredOnRecurringAndNonrecurringBasisValuationTechniquesTable" xlink:type="arc" order="10" xbrldt:closed="true" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_MeasurementInputTypeAxis" xlink:label="loc_us-gaapMeasurementInputTypeAxis" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_us-gaapFairValueAssetsAndLiabilitiesMeasuredOnRecurringAndNonrecurringBasisValuationTechniquesTable" xlink:to="loc_us-gaapMeasurementInputTypeAxis" xlink:type="arc" order="1" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_MeasurementInputTypeDomain" xlink:label="loc_us-gaapMeasurementInputTypeDomain" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_us-gaapMeasurementInputTypeAxis" xlink:to="loc_us-gaapMeasurementInputTypeDomain" xlink:type="arc" order="20" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_MeasurementInputTypeDomain" xlink:label="loc_us-gaapMeasurementInputTypeDomain_10" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xbrldt:contextElement="segment" xlink:from="loc_us-gaapMeasurementInputTypeAxis" xlink:to="loc_us-gaapMeasurementInputTypeDomain_10" xlink:type="arc" order="30" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_MeasurementInputPriceVolatilityMember" xlink:label="loc_us-gaapMeasurementInputPriceVolatilityMember_0" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapMeasurementInputTypeDomain" xlink:to="loc_us-gaapMeasurementInputPriceVolatilityMember_0" xlink:type="arc" order="1" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_MeasurementInputRiskFreeInterestRateMember" xlink:label="loc_us-gaapMeasurementInputRiskFreeInterestRateMember_20" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapMeasurementInputTypeDomain" xlink:to="loc_us-gaapMeasurementInputRiskFreeInterestRateMember_20" xlink:type="arc" order="21" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_MeasurementInputExpectedDividendRateMember" xlink:label="loc_us-gaapMeasurementInputExpectedDividendRateMember_40" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapMeasurementInputTypeDomain" xlink:to="loc_us-gaapMeasurementInputExpectedDividendRateMember_40" xlink:type="arc" order="41" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_MeasurementInputExpectedTermMember" xlink:label="loc_us-gaapMeasurementInputExpectedTermMember_60" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapMeasurementInputTypeDomain" xlink:to="loc_us-gaapMeasurementInputExpectedTermMember_60" xlink:type="arc" order="61" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_MeasurementInputExercisePriceMember" xlink:label="loc_us-gaapMeasurementInputExercisePriceMember_80" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapMeasurementInputTypeDomain" xlink:to="loc_us-gaapMeasurementInputExercisePriceMember_80" xlink:type="arc" order="81" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_MeasurementInputSharePriceMember" xlink:label="loc_us-gaapMeasurementInputSharePriceMember_100" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapMeasurementInputTypeDomain" xlink:to="loc_us-gaapMeasurementInputSharePriceMember_100" xlink:type="arc" order="101" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_StatementEquityComponentsAxis" xlink:label="loc_us-gaapStatementEquityComponentsAxis" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_us-gaapFairValueAssetsAndLiabilitiesMeasuredOnRecurringAndNonrecurringBasisValuationTechniquesTable" xlink:to="loc_us-gaapStatementEquityComponentsAxis" xlink:type="arc" order="2" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_EquityComponentDomain" xlink:label="loc_us-gaapEquityComponentDomain" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_us-gaapStatementEquityComponentsAxis" xlink:to="loc_us-gaapEquityComponentDomain" xlink:type="arc" order="20" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_EquityComponentDomain" xlink:label="loc_us-gaapEquityComponentDomain_10" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xbrldt:contextElement="segment" xlink:from="loc_us-gaapStatementEquityComponentsAxis" xlink:to="loc_us-gaapEquityComponentDomain_10" xlink:type="arc" order="30" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_WarrantMember" xlink:label="loc_us-gaapWarrantMember_0" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapEquityComponentDomain" xlink:to="loc_us-gaapWarrantMember_0" xlink:type="arc" order="2" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_WarrantsAndRightsOutstandingMeasurementInput" xlink:label="loc_us-gaapWarrantsAndRightsOutstandingMeasurementInput_140" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapFairValueAssetsAndLiabilitiesMeasuredOnRecurringAndNonrecurringBasisValuationTechniquesLineItems" xlink:to="loc_us-gaapWarrantsAndRightsOutstandingMeasurementInput_140" xlink:type="arc" order="0" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_WarrantsAndRightsOutstandingTerm" xlink:label="loc_us-gaapWarrantsAndRightsOutstandingTerm_140" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapFairValueAssetsAndLiabilitiesMeasuredOnRecurringAndNonrecurringBasisValuationTechniquesLineItems" xlink:to="loc_us-gaapWarrantsAndRightsOutstandingTerm_140" xlink:type="arc" order="1" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_FairValueAdjustmentOfWarrants" xlink:label="loc_us-gaapFairValueAdjustmentOfWarrants_140" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapFairValueAssetsAndLiabilitiesMeasuredOnRecurringAndNonrecurringBasisValuationTechniquesLineItems" xlink:to="loc_us-gaapFairValueAdjustmentOfWarrants_140" xlink:type="arc" order="2" />
    </link:definitionLink>
    <link:definitionLink xlink:type="extended" xlink:role="http://n2off.com/role/CommitmentAndContingentLiabilitiesDetailsNarrative" xlink:title="999073 - Disclosure - COMMITMENT AND CONTINGENT LIABILITIES (Details Narrative)">
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_StatementLineItems" xlink:label="loc_us-gaapStatementLineItems" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_StatementTable" xlink:label="loc_us-gaapStatementTable" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xbrldt:contextElement="segment" xlink:from="loc_us-gaapStatementLineItems" xlink:to="loc_us-gaapStatementTable" xlink:type="arc" order="10" xbrldt:closed="true" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.sec.gov/dei/2024/dei-2024.xsd#dei_LegalEntityAxis" xlink:label="loc_deiLegalEntityAxis" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_us-gaapStatementTable" xlink:to="loc_deiLegalEntityAxis" xlink:type="arc" order="1" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.sec.gov/dei/2024/dei-2024.xsd#dei_EntityDomain" xlink:label="loc_deiEntityDomain" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_deiLegalEntityAxis" xlink:to="loc_deiEntityDomain" xlink:type="arc" order="20" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.sec.gov/dei/2024/dei-2024.xsd#dei_EntityDomain" xlink:label="loc_deiEntityDomain_10" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xbrldt:contextElement="segment" xlink:from="loc_deiLegalEntityAxis" xlink:to="loc_deiEntityDomain_10" xlink:type="arc" order="30" />
      <link:loc xlink:type="locator" xlink:href="nito-20241231.xsd#NITO_IsraeliInnovationAuthorityMember" xlink:label="loc_NITOIsraeliInnovationAuthorityMember_0" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_deiEntityDomain" xlink:to="loc_NITOIsraeliInnovationAuthorityMember_0" xlink:type="arc" order="1" />
      <link:loc xlink:type="locator" xlink:href="nito-20241231.xsd#NITO_EarthboundTechnologiesLLCMember" xlink:label="loc_NITOEarthboundTechnologiesLLCMember_30" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_deiEntityDomain" xlink:to="loc_NITOEarthboundTechnologiesLLCMember_30" xlink:type="arc" order="32" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_TypeOfArrangementAxis" xlink:label="loc_us-gaapTypeOfArrangementAxis" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_us-gaapStatementTable" xlink:to="loc_us-gaapTypeOfArrangementAxis" xlink:type="arc" order="2" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_ArrangementsAndNonarrangementTransactionsMember" xlink:label="loc_us-gaapArrangementsAndNonarrangementTransactionsMember" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_us-gaapTypeOfArrangementAxis" xlink:to="loc_us-gaapArrangementsAndNonarrangementTransactionsMember" xlink:type="arc" order="20" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_ArrangementsAndNonarrangementTransactionsMember" xlink:label="loc_us-gaapArrangementsAndNonarrangementTransactionsMember_10" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xbrldt:contextElement="segment" xlink:from="loc_us-gaapTypeOfArrangementAxis" xlink:to="loc_us-gaapArrangementsAndNonarrangementTransactionsMember_10" xlink:type="arc" order="30" />
      <link:loc xlink:type="locator" xlink:href="nito-20241231.xsd#NITO_CommissionAgreementMember" xlink:label="loc_NITOCommissionAgreementMember_30" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapArrangementsAndNonarrangementTransactionsMember" xlink:to="loc_NITOCommissionAgreementMember_30" xlink:type="arc" order="31" />
      <link:loc xlink:type="locator" xlink:href="nito-20241231.xsd#NITO_ConsultingAgreementMember" xlink:label="loc_NITOConsultingAgreementMember_80" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapArrangementsAndNonarrangementTransactionsMember" xlink:to="loc_NITOConsultingAgreementMember_80" xlink:type="arc" order="82" />
      <link:loc xlink:type="locator" xlink:href="nito-20241231.xsd#NITO_FacilityAgreementMember" xlink:label="loc_NITOFacilityAgreementMember_180" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapArrangementsAndNonarrangementTransactionsMember" xlink:to="loc_NITOFacilityAgreementMember_180" xlink:type="arc" order="181" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/srt/2024/elts/srt-2024.xsd#srt_TitleOfIndividualAxis" xlink:label="loc_srtTitleOfIndividualAxis" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_us-gaapStatementTable" xlink:to="loc_srtTitleOfIndividualAxis" xlink:type="arc" order="3" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/srt/2024/elts/srt-2024.xsd#srt_TitleOfIndividualWithRelationshipToEntityDomain" xlink:label="loc_srtTitleOfIndividualWithRelationshipToEntityDomain" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_srtTitleOfIndividualAxis" xlink:to="loc_srtTitleOfIndividualWithRelationshipToEntityDomain" xlink:type="arc" order="20" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/srt/2024/elts/srt-2024.xsd#srt_TitleOfIndividualWithRelationshipToEntityDomain" xlink:label="loc_srtTitleOfIndividualWithRelationshipToEntityDomain_10" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xbrldt:contextElement="segment" xlink:from="loc_srtTitleOfIndividualAxis" xlink:to="loc_srtTitleOfIndividualWithRelationshipToEntityDomain_10" xlink:type="arc" order="30" />
      <link:loc xlink:type="locator" xlink:href="nito-20241231.xsd#NITO_IntroducedPartiesMember" xlink:label="loc_NITOIntroducedPartiesMember_30" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_srtTitleOfIndividualWithRelationshipToEntityDomain" xlink:to="loc_NITOIntroducedPartiesMember_30" xlink:type="arc" order="33" />
      <link:loc xlink:type="locator" xlink:href="nito-20241231.xsd#NITO_ConsultantOneMember" xlink:label="loc_NITOConsultantOneMember_80" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_srtTitleOfIndividualWithRelationshipToEntityDomain" xlink:to="loc_NITOConsultantOneMember_80" xlink:type="arc" order="81" />
      <link:loc xlink:type="locator" xlink:href="nito-20241231.xsd#NITO_ConsultantTwoMember" xlink:label="loc_NITOConsultantTwoMember_90" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_srtTitleOfIndividualWithRelationshipToEntityDomain" xlink:to="loc_NITOConsultantTwoMember_90" xlink:type="arc" order="91" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/srt/2024/elts/srt-2024.xsd#srt_ChiefExecutiveOfficerMember" xlink:label="loc_srtChiefExecutiveOfficerMember_100" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_srtTitleOfIndividualWithRelationshipToEntityDomain" xlink:to="loc_srtChiefExecutiveOfficerMember_100" xlink:type="arc" order="101" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/srt/2024/elts/srt-2024.xsd#srt_BoardOfDirectorsChairmanMember" xlink:label="loc_srtBoardOfDirectorsChairmanMember_110" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_srtTitleOfIndividualWithRelationshipToEntityDomain" xlink:to="loc_srtBoardOfDirectorsChairmanMember_110" xlink:type="arc" order="111" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/srt/2024/elts/srt-2024.xsd#srt_StatementScenarioAxis" xlink:label="loc_srtStatementScenarioAxis" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_us-gaapStatementTable" xlink:to="loc_srtStatementScenarioAxis" xlink:type="arc" order="4" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/srt/2024/elts/srt-2024.xsd#srt_ScenarioUnspecifiedDomain" xlink:label="loc_srtScenarioUnspecifiedDomain" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_srtStatementScenarioAxis" xlink:to="loc_srtScenarioUnspecifiedDomain" xlink:type="arc" order="20" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/srt/2024/elts/srt-2024.xsd#srt_ScenarioUnspecifiedDomain" xlink:label="loc_srtScenarioUnspecifiedDomain_10" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xbrldt:contextElement="segment" xlink:from="loc_srtStatementScenarioAxis" xlink:to="loc_srtScenarioUnspecifiedDomain_10" xlink:type="arc" order="30" />
      <link:loc xlink:type="locator" xlink:href="nito-20241231.xsd#NITO_ExceedsInNetRevenueMember" xlink:label="loc_NITOExceedsInNetRevenueMember_40" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_srtScenarioUnspecifiedDomain" xlink:to="loc_NITOExceedsInNetRevenueMember_40" xlink:type="arc" order="44" />
      <link:loc xlink:type="locator" xlink:href="nito-20241231.xsd#NITO_OccuranceOfAdditionalEventsInAgreementMember" xlink:label="loc_NITOOccuranceOfAdditionalEventsInAgreementMember_60" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_srtScenarioUnspecifiedDomain" xlink:to="loc_NITOOccuranceOfAdditionalEventsInAgreementMember_60" xlink:type="arc" order="64" />
      <link:loc xlink:type="locator" xlink:href="nito-20241231.xsd#NITO_RoyaltyDescription" xlink:label="loc_NITORoyaltyDescription_220" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapStatementLineItems" xlink:to="loc_NITORoyaltyDescription_220" xlink:type="arc" order="0" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_RoyaltyGuaranteesCommitmentsAmount" xlink:label="loc_us-gaapRoyaltyGuaranteesCommitmentsAmount_220" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapStatementLineItems" xlink:to="loc_us-gaapRoyaltyGuaranteesCommitmentsAmount_220" xlink:type="arc" order="1" />
      <link:loc xlink:type="locator" xlink:href="nito-20241231.xsd#NITO_PercentageOfNetRevenues" xlink:label="loc_NITOPercentageOfNetRevenues_220" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapStatementLineItems" xlink:to="loc_NITOPercentageOfNetRevenues_220" xlink:type="arc" order="2" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_Revenues" xlink:label="loc_us-gaapRevenues_220" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapStatementLineItems" xlink:to="loc_us-gaapRevenues_220" xlink:type="arc" order="3" />
      <link:loc xlink:type="locator" xlink:href="nito-20241231.xsd#NITO_MaximumCompensationPercentageOfGrossProfit" xlink:label="loc_NITOMaximumCompensationPercentageOfGrossProfit_220" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapStatementLineItems" xlink:to="loc_NITOMaximumCompensationPercentageOfGrossProfit_220" xlink:type="arc" order="4" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_StockRepurchasedDuringPeriodShares" xlink:label="loc_us-gaapStockRepurchasedDuringPeriodShares_220" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapStatementLineItems" xlink:to="loc_us-gaapStockRepurchasedDuringPeriodShares_220" xlink:type="arc" order="5" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_SharesIssuedPricePerShare" xlink:label="loc_us-gaapSharesIssuedPricePerShare_220" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapStatementLineItems" xlink:to="loc_us-gaapSharesIssuedPricePerShare_220" xlink:type="arc" order="6" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_DebtInstrumentPeriodicPayment" xlink:label="loc_us-gaapDebtInstrumentPeriodicPayment_220" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapStatementLineItems" xlink:to="loc_us-gaapDebtInstrumentPeriodicPayment_220" xlink:type="arc" order="7" />
      <link:loc xlink:type="locator" xlink:href="nito-20241231.xsd#NITO_PercentageOfConsultants" xlink:label="loc_NITOPercentageOfConsultants_220" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapStatementLineItems" xlink:to="loc_NITOPercentageOfConsultants_220" xlink:type="arc" order="8" />
      <link:loc xlink:type="locator" xlink:href="nito-20241231.xsd#NITO_ReimbursementExpenses" xlink:label="loc_NITOReimbursementExpenses_220" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapStatementLineItems" xlink:to="loc_NITOReimbursementExpenses_220" xlink:type="arc" order="9" />
      <link:loc xlink:type="locator" xlink:href="nito-20241231.xsd#NITO_SuccessFeesGrossProceedsPercentage" xlink:label="loc_NITOSuccessFeesGrossProceedsPercentage_220" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapStatementLineItems" xlink:to="loc_NITOSuccessFeesGrossProceedsPercentage_220" xlink:type="arc" order="10" />
      <link:loc xlink:type="locator" xlink:href="nito-20241231.xsd#NITO_SuccessFeesPercentage" xlink:label="loc_NITOSuccessFeesPercentage_220" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapStatementLineItems" xlink:to="loc_NITOSuccessFeesPercentage_220" xlink:type="arc" order="11" />
      <link:loc xlink:type="locator" xlink:href="nito-20241231.xsd#NITO_SuccessFees" xlink:label="loc_NITOSuccessFees_220" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapStatementLineItems" xlink:to="loc_NITOSuccessFees_220" xlink:type="arc" order="12" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_LineOfCreditFacilityMaximumBorrowingCapacity" xlink:label="loc_us-gaapLineOfCreditFacilityMaximumBorrowingCapacity_220" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapStatementLineItems" xlink:to="loc_us-gaapLineOfCreditFacilityMaximumBorrowingCapacity_220" xlink:type="arc" order="13" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_LineOfCreditFacilityCurrentBorrowingCapacity" xlink:label="loc_us-gaapLineOfCreditFacilityCurrentBorrowingCapacity_220" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapStatementLineItems" xlink:to="loc_us-gaapLineOfCreditFacilityCurrentBorrowingCapacity_220" xlink:type="arc" order="14" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_LineOfCreditFacilityRemainingBorrowingCapacity" xlink:label="loc_us-gaapLineOfCreditFacilityRemainingBorrowingCapacity_220" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapStatementLineItems" xlink:to="loc_us-gaapLineOfCreditFacilityRemainingBorrowingCapacity_220" xlink:type="arc" order="15" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_LineOfCreditFacilityInterestRateDuringPeriod" xlink:label="loc_us-gaapLineOfCreditFacilityInterestRateDuringPeriod_220" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapStatementLineItems" xlink:to="loc_us-gaapLineOfCreditFacilityInterestRateDuringPeriod_220" xlink:type="arc" order="16" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_LineOfCreditFacilityDecreaseForgiveness1" xlink:label="loc_us-gaapLineOfCreditFacilityDecreaseForgiveness1_220" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapStatementLineItems" xlink:to="loc_us-gaapLineOfCreditFacilityDecreaseForgiveness1_220" xlink:type="arc" order="17" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_LineOfCreditFacilityExpirationPeriod" xlink:label="loc_us-gaapLineOfCreditFacilityExpirationPeriod_220" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapStatementLineItems" xlink:to="loc_us-gaapLineOfCreditFacilityExpirationPeriod_220" xlink:type="arc" order="18" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_LineOfCreditFacilityInterestRateAtPeriodEnd" xlink:label="loc_us-gaapLineOfCreditFacilityInterestRateAtPeriodEnd_220" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapStatementLineItems" xlink:to="loc_us-gaapLineOfCreditFacilityInterestRateAtPeriodEnd_220" xlink:type="arc" order="19" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_WarrantsAndRightsOutstandingTerm" xlink:label="loc_us-gaapWarrantsAndRightsOutstandingTerm_220" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapStatementLineItems" xlink:to="loc_us-gaapWarrantsAndRightsOutstandingTerm_220" xlink:type="arc" order="20" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_ClassOfWarrantOrRightOutstanding" xlink:label="loc_us-gaapClassOfWarrantOrRightOutstanding_220" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapStatementLineItems" xlink:to="loc_us-gaapClassOfWarrantOrRightOutstanding_220" xlink:type="arc" order="21" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_ClassOfWarrantOrRightExercisePriceOfWarrantsOrRights1" xlink:label="loc_us-gaapClassOfWarrantOrRightExercisePriceOfWarrantsOrRights1_220" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapStatementLineItems" xlink:to="loc_us-gaapClassOfWarrantOrRightExercisePriceOfWarrantsOrRights1_220" xlink:type="arc" order="22" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_LongTermLineOfCredit" xlink:label="loc_us-gaapLongTermLineOfCredit_220" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapStatementLineItems" xlink:to="loc_us-gaapLongTermLineOfCredit_220" xlink:type="arc" order="23" />
    </link:definitionLink>
    <link:definitionLink xlink:type="extended" xlink:role="http://n2off.com/role/ScheduleOfOperatingLeaseCostDetails" xlink:title="999074 - Disclosure - SCHEDULE OF OPERATING LEASE COST (Details)" />
    <link:definitionLink xlink:type="extended" xlink:role="http://n2off.com/role/ScheduleOfSupplementalCashFlowInformationRelatedToOperatingLeaseDetails" xlink:title="999075 - Disclosure - SCHEDULE OF SUPPLEMENTAL CASH FLOW INFORMATION RELATED TO OPERATING LEASE (Details)" />
    <link:definitionLink xlink:type="extended" xlink:role="http://n2off.com/role/ScheduleOfSupplementalBalanceSheetInformationRelatedToOperatingLeasesDetails" xlink:title="999076 - Disclosure - SCHEDULE OF SUPPLEMENTAL BALANCE SHEET INFORMATION RELATED TO OPERATING LEASES (Details)" />
    <link:definitionLink xlink:type="extended" xlink:role="http://n2off.com/role/ScheduleOfMinimumLeasePaymentsUnderNon-CancelableLeasesDetails" xlink:title="999077 - Disclosure - SCHEDULE OF MINIMUM LEASE PAYMENTS UNDER NON- CANCELABLE LEASES (Details)" />
    <link:definitionLink xlink:type="extended" xlink:role="http://n2off.com/role/LeasesDetailsNarrative" xlink:title="999078 - Disclosure - LEASES (Details Narrative)">
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_StatementLineItems" xlink:label="loc_us-gaapStatementLineItems" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_StatementTable" xlink:label="loc_us-gaapStatementTable" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xbrldt:contextElement="segment" xlink:from="loc_us-gaapStatementLineItems" xlink:to="loc_us-gaapStatementTable" xlink:type="arc" order="10" xbrldt:closed="true" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/srt/2024/elts/srt-2024.xsd#srt_StatementGeographicalAxis" xlink:label="loc_srtStatementGeographicalAxis" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_us-gaapStatementTable" xlink:to="loc_srtStatementGeographicalAxis" xlink:type="arc" order="1" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/srt/2024/elts/srt-2024.xsd#srt_SegmentGeographicalDomain" xlink:label="loc_srtSegmentGeographicalDomain" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_srtStatementGeographicalAxis" xlink:to="loc_srtSegmentGeographicalDomain" xlink:type="arc" order="20" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/srt/2024/elts/srt-2024.xsd#srt_SegmentGeographicalDomain" xlink:label="loc_srtSegmentGeographicalDomain_10" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xbrldt:contextElement="segment" xlink:from="loc_srtStatementGeographicalAxis" xlink:to="loc_srtSegmentGeographicalDomain_10" xlink:type="arc" order="30" />
      <link:loc xlink:type="locator" xlink:href="nito-20241231.xsd#NITO_NeveYarakMember" xlink:label="loc_NITONeveYarakMember_0" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_srtSegmentGeographicalDomain" xlink:to="loc_NITONeveYarakMember_0" xlink:type="arc" order="1" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_AwardTypeAxis" xlink:label="loc_us-gaapAwardTypeAxis" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_us-gaapStatementTable" xlink:to="loc_us-gaapAwardTypeAxis" xlink:type="arc" order="2" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain" xlink:label="loc_us-gaapShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_us-gaapAwardTypeAxis" xlink:to="loc_us-gaapShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain" xlink:type="arc" order="20" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain" xlink:label="loc_us-gaapShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain_10" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xbrldt:contextElement="segment" xlink:from="loc_us-gaapAwardTypeAxis" xlink:to="loc_us-gaapShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain_10" xlink:type="arc" order="30" />
      <link:loc xlink:type="locator" xlink:href="nito-20241231.xsd#NITO_FirstOptionPeriodMember" xlink:label="loc_NITOFirstOptionPeriodMember_20" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain" xlink:to="loc_NITOFirstOptionPeriodMember_20" xlink:type="arc" order="22" />
      <link:loc xlink:type="locator" xlink:href="nito-20241231.xsd#NITO_SecondOptionPeriodMember" xlink:label="loc_NITOSecondOptionPeriodMember_30" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain" xlink:to="loc_NITOSecondOptionPeriodMember_30" xlink:type="arc" order="32" />
      <link:loc xlink:type="locator" xlink:href="nito-20241231.xsd#NITO_AdditionalOptionPeriodMember" xlink:label="loc_NITOAdditionalOptionPeriodMember_40" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain" xlink:to="loc_NITOAdditionalOptionPeriodMember_40" xlink:type="arc" order="42" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_TypeOfArrangementAxis" xlink:label="loc_us-gaapTypeOfArrangementAxis" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_us-gaapStatementTable" xlink:to="loc_us-gaapTypeOfArrangementAxis" xlink:type="arc" order="3" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_ArrangementsAndNonarrangementTransactionsMember" xlink:label="loc_us-gaapArrangementsAndNonarrangementTransactionsMember" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_us-gaapTypeOfArrangementAxis" xlink:to="loc_us-gaapArrangementsAndNonarrangementTransactionsMember" xlink:type="arc" order="20" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_ArrangementsAndNonarrangementTransactionsMember" xlink:label="loc_us-gaapArrangementsAndNonarrangementTransactionsMember_10" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xbrldt:contextElement="segment" xlink:from="loc_us-gaapTypeOfArrangementAxis" xlink:to="loc_us-gaapArrangementsAndNonarrangementTransactionsMember_10" xlink:type="arc" order="30" />
      <link:loc xlink:type="locator" xlink:href="nito-20241231.xsd#NITO_LeaseAgreementMember" xlink:label="loc_NITOLeaseAgreementMember_50" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapArrangementsAndNonarrangementTransactionsMember" xlink:to="loc_NITOLeaseAgreementMember_50" xlink:type="arc" order="51" />
      <link:loc xlink:type="locator" xlink:href="nito-20241231.xsd#NITO_CarRentalAgreementMember" xlink:label="loc_NITOCarRentalAgreementMember_60" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapArrangementsAndNonarrangementTransactionsMember" xlink:to="loc_NITOCarRentalAgreementMember_60" xlink:type="arc" order="61" />
      <link:loc xlink:type="locator" xlink:href="nito-20241231.xsd#NITO_MiamiLeaseAgreementMember" xlink:label="loc_NITOMiamiLeaseAgreementMember_80" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapArrangementsAndNonarrangementTransactionsMember" xlink:to="loc_NITOMiamiLeaseAgreementMember_80" xlink:type="arc" order="81" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_LesseeOperatingLeaseTermOfContract" xlink:label="loc_us-gaapLesseeOperatingLeaseTermOfContract_150" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapStatementLineItems" xlink:to="loc_us-gaapLesseeOperatingLeaseTermOfContract_150" xlink:type="arc" order="0" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_PaymentsForRent" xlink:label="loc_us-gaapPaymentsForRent_150" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapStatementLineItems" xlink:to="loc_us-gaapPaymentsForRent_150" xlink:type="arc" order="1" />
      <link:loc xlink:type="locator" xlink:href="nito-20241231.xsd#NITO_LesseeOperatingAdditionalLeaseTermOfContract" xlink:label="loc_NITOLesseeOperatingAdditionalLeaseTermOfContract_150" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapStatementLineItems" xlink:to="loc_NITOLesseeOperatingAdditionalLeaseTermOfContract_150" xlink:type="arc" order="2" />
      <link:loc xlink:type="locator" xlink:href="nito-20241231.xsd#NITO_AdditionalPaymentsForRent" xlink:label="loc_NITOAdditionalPaymentsForRent_150" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapStatementLineItems" xlink:to="loc_NITOAdditionalPaymentsForRent_150" xlink:type="arc" order="3" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_OperatingLeaseRightOfUseAsset" xlink:label="loc_us-gaapOperatingLeaseRightOfUseAsset_150" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapStatementLineItems" xlink:to="loc_us-gaapOperatingLeaseRightOfUseAsset_150" xlink:type="arc" order="4" />
    </link:definitionLink>
    <link:definitionLink xlink:type="extended" xlink:role="http://n2off.com/role/ScheduleOfTransactionsAndBalancesWithRelatedPartiesDetailsParenthetical" xlink:title="999079 - Disclosure - SCHEDULE OF TRANSACTIONS AND BALANCES WITH RELATED PARTIES (Details) (Parenthetical)">
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_CollaborativeArrangementsAndNoncollaborativeArrangementTransactionsLineItems" xlink:label="loc_us-gaapCollaborativeArrangementsAndNoncollaborativeArrangementTransactionsLineItems" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_ScheduleOfCollaborativeArrangementsAndNoncollaborativeArrangementTransactionsTable" xlink:label="loc_us-gaapScheduleOfCollaborativeArrangementsAndNoncollaborativeArrangementTransactionsTable" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xbrldt:contextElement="segment" xlink:from="loc_us-gaapCollaborativeArrangementsAndNoncollaborativeArrangementTransactionsLineItems" xlink:to="loc_us-gaapScheduleOfCollaborativeArrangementsAndNoncollaborativeArrangementTransactionsTable" xlink:type="arc" order="10" xbrldt:closed="true" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_TypeOfArrangementAxis" xlink:label="loc_us-gaapTypeOfArrangementAxis" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_us-gaapScheduleOfCollaborativeArrangementsAndNoncollaborativeArrangementTransactionsTable" xlink:to="loc_us-gaapTypeOfArrangementAxis" xlink:type="arc" order="1" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_ArrangementsAndNonarrangementTransactionsMember" xlink:label="loc_us-gaapArrangementsAndNonarrangementTransactionsMember" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_us-gaapTypeOfArrangementAxis" xlink:to="loc_us-gaapArrangementsAndNonarrangementTransactionsMember" xlink:type="arc" order="20" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_ArrangementsAndNonarrangementTransactionsMember" xlink:label="loc_us-gaapArrangementsAndNonarrangementTransactionsMember_10" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xbrldt:contextElement="segment" xlink:from="loc_us-gaapTypeOfArrangementAxis" xlink:to="loc_us-gaapArrangementsAndNonarrangementTransactionsMember_10" xlink:type="arc" order="30" />
      <link:loc xlink:type="locator" xlink:href="nito-20241231.xsd#NITO_ConsultingAgreementMember" xlink:label="loc_NITOConsultingAgreementMember_0" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapArrangementsAndNonarrangementTransactionsMember" xlink:to="loc_NITOConsultingAgreementMember_0" xlink:type="arc" order="1" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_StockIssuedDuringPeriodValueRestrictedStockAwardGross" xlink:label="loc_us-gaapStockIssuedDuringPeriodValueRestrictedStockAwardGross_20" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapCollaborativeArrangementsAndNoncollaborativeArrangementTransactionsLineItems" xlink:to="loc_us-gaapStockIssuedDuringPeriodValueRestrictedStockAwardGross_20" xlink:type="arc" order="0" />
    </link:definitionLink>
    <link:definitionLink xlink:type="extended" xlink:role="http://n2off.com/role/StockholdersEquityDetailsNarrative" xlink:title="999080 - Disclosure - STOCKHOLDERS&#8217; EQUITY (Details Narrative)">
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_StatementLineItems" xlink:label="loc_us-gaapStatementLineItems" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_StatementTable" xlink:label="loc_us-gaapStatementTable" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xbrldt:contextElement="segment" xlink:from="loc_us-gaapStatementLineItems" xlink:to="loc_us-gaapStatementTable" xlink:type="arc" order="10" xbrldt:closed="true" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_TypeOfArrangementAxis" xlink:label="loc_us-gaapTypeOfArrangementAxis" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_us-gaapStatementTable" xlink:to="loc_us-gaapTypeOfArrangementAxis" xlink:type="arc" order="1" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_ArrangementsAndNonarrangementTransactionsMember" xlink:label="loc_us-gaapArrangementsAndNonarrangementTransactionsMember" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_us-gaapTypeOfArrangementAxis" xlink:to="loc_us-gaapArrangementsAndNonarrangementTransactionsMember" xlink:type="arc" order="20" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_ArrangementsAndNonarrangementTransactionsMember" xlink:label="loc_us-gaapArrangementsAndNonarrangementTransactionsMember_10" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xbrldt:contextElement="segment" xlink:from="loc_us-gaapTypeOfArrangementAxis" xlink:to="loc_us-gaapArrangementsAndNonarrangementTransactionsMember_10" xlink:type="arc" order="30" />
      <link:loc xlink:type="locator" xlink:href="nito-20241231.xsd#NITO_ConsultingAgreementMember" xlink:label="loc_NITOConsultingAgreementMember_0" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapArrangementsAndNonarrangementTransactionsMember" xlink:to="loc_NITOConsultingAgreementMember_0" xlink:type="arc" order="1" />
      <link:loc xlink:type="locator" xlink:href="nito-20241231.xsd#NITO_TerminationAgreementsMember" xlink:label="loc_NITOTerminationAgreementsMember_50" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapArrangementsAndNonarrangementTransactionsMember" xlink:to="loc_NITOTerminationAgreementsMember_50" xlink:type="arc" order="51" />
      <link:loc xlink:type="locator" xlink:href="nito-20241231.xsd#NITO_InvestorRelationsAgreementMember" xlink:label="loc_NITOInvestorRelationsAgreementMember_60" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapArrangementsAndNonarrangementTransactionsMember" xlink:to="loc_NITOInvestorRelationsAgreementMember_60" xlink:type="arc" order="62" />
      <link:loc xlink:type="locator" xlink:href="nito-20241231.xsd#NITO_ServiceAgreementMember" xlink:label="loc_NITOServiceAgreementMember_80" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapArrangementsAndNonarrangementTransactionsMember" xlink:to="loc_NITOServiceAgreementMember_80" xlink:type="arc" order="82" />
      <link:loc xlink:type="locator" xlink:href="nito-20241231.xsd#NITO_ConsultingServicesMember" xlink:label="loc_NITOConsultingServicesMember_190" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapArrangementsAndNonarrangementTransactionsMember" xlink:to="loc_NITOConsultingServicesMember_190" xlink:type="arc" order="192" />
      <link:loc xlink:type="locator" xlink:href="nito-20241231.xsd#NITO_ConsultingAgreementsMember" xlink:label="loc_NITOConsultingAgreementsMember_220" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapArrangementsAndNonarrangementTransactionsMember" xlink:to="loc_NITOConsultingAgreementsMember_220" xlink:type="arc" order="221" />
      <link:loc xlink:type="locator" xlink:href="nito-20241231.xsd#NITO_SecuritiesExchangeAgreementMember" xlink:label="loc_NITOSecuritiesExchangeAgreementMember_560" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapArrangementsAndNonarrangementTransactionsMember" xlink:to="loc_NITOSecuritiesExchangeAgreementMember_560" xlink:type="arc" order="562" />
      <link:loc xlink:type="locator" xlink:href="nito-20241231.xsd#NITO_RetainerLegalServicesAgreementMember" xlink:label="loc_NITORetainerLegalServicesAgreementMember_780" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapArrangementsAndNonarrangementTransactionsMember" xlink:to="loc_NITORetainerLegalServicesAgreementMember_780" xlink:type="arc" order="781" />
      <link:loc xlink:type="locator" xlink:href="nito-20241231.xsd#NITO_StandbyEquityPurchaseAgreementMember" xlink:label="loc_NITOStandbyEquityPurchaseAgreementMember_850" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapArrangementsAndNonarrangementTransactionsMember" xlink:to="loc_NITOStandbyEquityPurchaseAgreementMember_850" xlink:type="arc" order="851" />
      <link:loc xlink:type="locator" xlink:href="nito-20241231.xsd#NITO_PurchaseAgreementMember" xlink:label="loc_NITOPurchaseAgreementMember_930" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapArrangementsAndNonarrangementTransactionsMember" xlink:to="loc_NITOPurchaseAgreementMember_930" xlink:type="arc" order="931" />
      <link:loc xlink:type="locator" xlink:href="nito-20241231.xsd#NITO_StandbyEquityPurchaseAgreementTwoMember" xlink:label="loc_NITOStandbyEquityPurchaseAgreementTwoMember_1080" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapArrangementsAndNonarrangementTransactionsMember" xlink:to="loc_NITOStandbyEquityPurchaseAgreementTwoMember_1080" xlink:type="arc" order="1081" />
      <link:loc xlink:type="locator" xlink:href="nito-20241231.xsd#NITO_ExchangeAgreementMember" xlink:label="loc_NITOExchangeAgreementMember_1090" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapArrangementsAndNonarrangementTransactionsMember" xlink:to="loc_NITOExchangeAgreementMember_1090" xlink:type="arc" order="1091" />
      <link:loc xlink:type="locator" xlink:href="nito-20241231.xsd#NITO_SecuritiesPurchaseAgreementMember" xlink:label="loc_NITOSecuritiesPurchaseAgreementMember_1220" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapArrangementsAndNonarrangementTransactionsMember" xlink:to="loc_NITOSecuritiesPurchaseAgreementMember_1220" xlink:type="arc" order="1221" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/srt/2024/elts/srt-2024.xsd#srt_TitleOfIndividualAxis" xlink:label="loc_srtTitleOfIndividualAxis" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_us-gaapStatementTable" xlink:to="loc_srtTitleOfIndividualAxis" xlink:type="arc" order="2" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/srt/2024/elts/srt-2024.xsd#srt_TitleOfIndividualWithRelationshipToEntityDomain" xlink:label="loc_srtTitleOfIndividualWithRelationshipToEntityDomain" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_srtTitleOfIndividualAxis" xlink:to="loc_srtTitleOfIndividualWithRelationshipToEntityDomain" xlink:type="arc" order="20" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/srt/2024/elts/srt-2024.xsd#srt_TitleOfIndividualWithRelationshipToEntityDomain" xlink:label="loc_srtTitleOfIndividualWithRelationshipToEntityDomain_10" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xbrldt:contextElement="segment" xlink:from="loc_srtTitleOfIndividualAxis" xlink:to="loc_srtTitleOfIndividualWithRelationshipToEntityDomain_10" xlink:type="arc" order="30" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/srt/2024/elts/srt-2024.xsd#srt_BoardOfDirectorsChairmanMember" xlink:label="loc_srtBoardOfDirectorsChairmanMember_30" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_srtTitleOfIndividualWithRelationshipToEntityDomain" xlink:to="loc_srtBoardOfDirectorsChairmanMember_30" xlink:type="arc" order="31" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/srt/2024/elts/srt-2024.xsd#srt_DirectorMember" xlink:label="loc_srtDirectorMember_40" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_srtTitleOfIndividualWithRelationshipToEntityDomain" xlink:to="loc_srtDirectorMember_40" xlink:type="arc" order="42" />
      <link:loc xlink:type="locator" xlink:href="nito-20241231.xsd#NITO_ConsultantMember" xlink:label="loc_NITOConsultantMember_60" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_srtTitleOfIndividualWithRelationshipToEntityDomain" xlink:to="loc_NITOConsultantMember_60" xlink:type="arc" order="61" />
      <link:loc xlink:type="locator" xlink:href="nito-20241231.xsd#NITO_TwoConsultantMember" xlink:label="loc_NITOTwoConsultantMember_230" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_srtTitleOfIndividualWithRelationshipToEntityDomain" xlink:to="loc_NITOTwoConsultantMember_230" xlink:type="arc" order="231" />
      <link:loc xlink:type="locator" xlink:href="nito-20241231.xsd#NITO_MrJoachimFuchsMember" xlink:label="loc_NITOMrJoachimFuchsMember_540" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_srtTitleOfIndividualWithRelationshipToEntityDomain" xlink:to="loc_NITOMrJoachimFuchsMember_540" xlink:type="arc" order="542" />
      <link:loc xlink:type="locator" xlink:href="nito-20241231.xsd#NITO_LegalAdvisorsMember" xlink:label="loc_NITOLegalAdvisorsMember_740" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_srtTitleOfIndividualWithRelationshipToEntityDomain" xlink:to="loc_NITOLegalAdvisorsMember_740" xlink:type="arc" order="742" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_InvestorMember" xlink:label="loc_us-gaapInvestorMember_1020" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_srtTitleOfIndividualWithRelationshipToEntityDomain" xlink:to="loc_us-gaapInvestorMember_1020" xlink:type="arc" order="1022" />
      <link:loc xlink:type="locator" xlink:href="nito-20241231.xsd#NITO_ThirdPartyMember" xlink:label="loc_NITOThirdPartyMember_1120" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_srtTitleOfIndividualWithRelationshipToEntityDomain" xlink:to="loc_NITOThirdPartyMember_1120" xlink:type="arc" order="1122" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/srt/2024/elts/srt-2024.xsd#srt_ExecutiveOfficerMember" xlink:label="loc_srtExecutiveOfficerMember_1170" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_srtTitleOfIndividualWithRelationshipToEntityDomain" xlink:to="loc_srtExecutiveOfficerMember_1170" xlink:type="arc" order="1171" />
      <link:loc xlink:type="locator" xlink:href="nito-20241231.xsd#NITO_ConsultantsMember" xlink:label="loc_NITOConsultantsMember_1180" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_srtTitleOfIndividualWithRelationshipToEntityDomain" xlink:to="loc_NITOConsultantsMember_1180" xlink:type="arc" order="1181" />
      <link:loc xlink:type="locator" xlink:href="nito-20241231.xsd#NITO_BoardOfDirectorsMember" xlink:label="loc_NITOBoardOfDirectorsMember_1210" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_srtTitleOfIndividualWithRelationshipToEntityDomain" xlink:to="loc_NITOBoardOfDirectorsMember_1210" xlink:type="arc" order="1211" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_StatementEquityComponentsAxis" xlink:label="loc_us-gaapStatementEquityComponentsAxis" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_us-gaapStatementTable" xlink:to="loc_us-gaapStatementEquityComponentsAxis" xlink:type="arc" order="3" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_EquityComponentDomain" xlink:label="loc_us-gaapEquityComponentDomain" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_us-gaapStatementEquityComponentsAxis" xlink:to="loc_us-gaapEquityComponentDomain" xlink:type="arc" order="20" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_EquityComponentDomain" xlink:label="loc_us-gaapEquityComponentDomain_10" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xbrldt:contextElement="segment" xlink:from="loc_us-gaapStatementEquityComponentsAxis" xlink:to="loc_us-gaapEquityComponentDomain_10" xlink:type="arc" order="30" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_CommonStockMember" xlink:label="loc_us-gaapCommonStockMember_40" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapEquityComponentDomain" xlink:to="loc_us-gaapCommonStockMember_40" xlink:type="arc" order="41" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_WarrantMember" xlink:label="loc_us-gaapWarrantMember_1250" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapEquityComponentDomain" xlink:to="loc_us-gaapWarrantMember_1250" xlink:type="arc" order="1251" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/srt/2024/elts/srt-2024.xsd#srt_StatementScenarioAxis" xlink:label="loc_srtStatementScenarioAxis" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_us-gaapStatementTable" xlink:to="loc_srtStatementScenarioAxis" xlink:type="arc" order="4" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/srt/2024/elts/srt-2024.xsd#srt_ScenarioUnspecifiedDomain" xlink:label="loc_srtScenarioUnspecifiedDomain" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_srtStatementScenarioAxis" xlink:to="loc_srtScenarioUnspecifiedDomain" xlink:type="arc" order="20" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/srt/2024/elts/srt-2024.xsd#srt_ScenarioUnspecifiedDomain" xlink:label="loc_srtScenarioUnspecifiedDomain_10" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xbrldt:contextElement="segment" xlink:from="loc_srtStatementScenarioAxis" xlink:to="loc_srtScenarioUnspecifiedDomain_10" xlink:type="arc" order="30" />
      <link:loc xlink:type="locator" xlink:href="nito-20241231.xsd#NITO_FirstQuarterTwentyTwentyThreeMember" xlink:label="loc_NITOFirstQuarterTwentyTwentyThreeMember_320" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_srtScenarioUnspecifiedDomain" xlink:to="loc_NITOFirstQuarterTwentyTwentyThreeMember_320" xlink:type="arc" order="323" />
      <link:loc xlink:type="locator" xlink:href="nito-20241231.xsd#NITO_EachQuarterTwentyTwentyThreeMember" xlink:label="loc_NITOEachQuarterTwentyTwentyThreeMember_330" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_srtScenarioUnspecifiedDomain" xlink:to="loc_NITOEachQuarterTwentyTwentyThreeMember_330" xlink:type="arc" order="333" />
      <link:loc xlink:type="locator" xlink:href="nito-20241231.xsd#NITO_TwoYearsMember" xlink:label="loc_NITOTwoYearsMember_390" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_srtScenarioUnspecifiedDomain" xlink:to="loc_NITOTwoYearsMember_390" xlink:type="arc" order="393" />
      <link:loc xlink:type="locator" xlink:href="nito-20241231.xsd#NITO_EighteenMonthsMember" xlink:label="loc_NITOEighteenMonthsMember_590" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_srtScenarioUnspecifiedDomain" xlink:to="loc_NITOEighteenMonthsMember_590" xlink:type="arc" order="593" />
      <link:loc xlink:type="locator" xlink:href="nito-20241231.xsd#NITO_EighteenMonthMember" xlink:label="loc_NITOEighteenMonthMember_610" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_srtScenarioUnspecifiedDomain" xlink:to="loc_NITOEighteenMonthMember_610" xlink:type="arc" order="613" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_AwardTypeAxis" xlink:label="loc_us-gaapAwardTypeAxis" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_us-gaapStatementTable" xlink:to="loc_us-gaapAwardTypeAxis" xlink:type="arc" order="5" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain" xlink:label="loc_us-gaapShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_us-gaapAwardTypeAxis" xlink:to="loc_us-gaapShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain" xlink:type="arc" order="20" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain" xlink:label="loc_us-gaapShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain_10" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xbrldt:contextElement="segment" xlink:from="loc_us-gaapAwardTypeAxis" xlink:to="loc_us-gaapShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain_10" xlink:type="arc" order="30" />
      <link:loc xlink:type="locator" xlink:href="nito-20241231.xsd#NITO_SixMonthsAnniversaryMember" xlink:label="loc_NITOSixMonthsAnniversaryMember_390" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain" xlink:to="loc_NITOSixMonthsAnniversaryMember_390" xlink:type="arc" order="391" />
      <link:loc xlink:type="locator" xlink:href="nito-20241231.xsd#NITO_NineMonthsAnniversaryMember" xlink:label="loc_NITONineMonthsAnniversaryMember_400" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain" xlink:to="loc_NITONineMonthsAnniversaryMember_400" xlink:type="arc" order="402" />
      <link:loc xlink:type="locator" xlink:href="nito-20241231.xsd#NITO_TwelveMonthsAnniversaryMember" xlink:label="loc_NITOTwelveMonthsAnniversaryMember_410" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain" xlink:to="loc_NITOTwelveMonthsAnniversaryMember_410" xlink:type="arc" order="411" />
      <link:loc xlink:type="locator" xlink:href="nito-20241231.xsd#NITO_ThreeMonthsMember" xlink:label="loc_NITOThreeMonthsMember_720" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain" xlink:to="loc_NITOThreeMonthsMember_720" xlink:type="arc" order="722" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.sec.gov/dei/2024/dei-2024.xsd#dei_LegalEntityAxis" xlink:label="loc_deiLegalEntityAxis" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_us-gaapStatementTable" xlink:to="loc_deiLegalEntityAxis" xlink:type="arc" order="6" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.sec.gov/dei/2024/dei-2024.xsd#dei_EntityDomain" xlink:label="loc_deiEntityDomain" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_deiLegalEntityAxis" xlink:to="loc_deiEntityDomain" xlink:type="arc" order="20" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.sec.gov/dei/2024/dei-2024.xsd#dei_EntityDomain" xlink:label="loc_deiEntityDomain_10" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xbrldt:contextElement="segment" xlink:from="loc_deiLegalEntityAxis" xlink:to="loc_deiEntityDomain_10" xlink:type="arc" order="30" />
      <link:loc xlink:type="locator" xlink:href="nito-20241231.xsd#NITO_EUAgritechInvestmentLtdMember" xlink:label="loc_NITOEUAgritechInvestmentLtdMember_500" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_deiEntityDomain" xlink:to="loc_NITOEUAgritechInvestmentLtdMember_500" xlink:type="arc" order="502" />
      <link:loc xlink:type="locator" xlink:href="nito-20241231.xsd#NITO_PlantifyFoodsIncMember" xlink:label="loc_NITOPlantifyFoodsIncMember_560" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_deiEntityDomain" xlink:to="loc_NITOPlantifyFoodsIncMember_560" xlink:type="arc" order="561" />
      <link:loc xlink:type="locator" xlink:href="nito-20241231.xsd#NITO_YAIIPNLtdMember" xlink:label="loc_NITOYAIIPNLtdMember_850" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_deiEntityDomain" xlink:to="loc_NITOYAIIPNLtdMember_850" xlink:type="arc" order="852" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/srt/2024/elts/srt-2024.xsd#srt_RangeAxis" xlink:label="loc_srtRangeAxis" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_us-gaapStatementTable" xlink:to="loc_srtRangeAxis" xlink:type="arc" order="7" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/srt/2024/elts/srt-2024.xsd#srt_RangeMember" xlink:label="loc_srtRangeMember" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_srtRangeAxis" xlink:to="loc_srtRangeMember" xlink:type="arc" order="20" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/srt/2024/elts/srt-2024.xsd#srt_RangeMember" xlink:label="loc_srtRangeMember_10" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xbrldt:contextElement="segment" xlink:from="loc_srtRangeAxis" xlink:to="loc_srtRangeMember_10" xlink:type="arc" order="30" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/srt/2024/elts/srt-2024.xsd#srt_MinimumMember" xlink:label="loc_srtMinimumMember_870" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_srtRangeMember" xlink:to="loc_srtMinimumMember_870" xlink:type="arc" order="871" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/srt/2024/elts/srt-2024.xsd#srt_MaximumMember" xlink:label="loc_srtMaximumMember_880" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_srtRangeMember" xlink:to="loc_srtMaximumMember_880" xlink:type="arc" order="881" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_DebtInstrumentAxis" xlink:label="loc_us-gaapDebtInstrumentAxis" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_us-gaapStatementTable" xlink:to="loc_us-gaapDebtInstrumentAxis" xlink:type="arc" order="8" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_DebtInstrumentNameDomain" xlink:label="loc_us-gaapDebtInstrumentNameDomain" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_us-gaapDebtInstrumentAxis" xlink:to="loc_us-gaapDebtInstrumentNameDomain" xlink:type="arc" order="20" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_DebtInstrumentNameDomain" xlink:label="loc_us-gaapDebtInstrumentNameDomain_10" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xbrldt:contextElement="segment" xlink:from="loc_us-gaapDebtInstrumentAxis" xlink:to="loc_us-gaapDebtInstrumentNameDomain_10" xlink:type="arc" order="30" />
      <link:loc xlink:type="locator" xlink:href="nito-20241231.xsd#NITO_PromissoryNoteMember" xlink:label="loc_NITOPromissoryNoteMember_1050" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapDebtInstrumentNameDomain" xlink:to="loc_NITOPromissoryNoteMember_1050" xlink:type="arc" order="1051" />
      <link:loc xlink:type="locator" xlink:href="nito-20241231.xsd#NITO_MonthlyConsultancyFee" xlink:label="loc_NITOMonthlyConsultancyFee_1270" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapStatementLineItems" xlink:to="loc_NITOMonthlyConsultancyFee_1270" xlink:type="arc" order="0" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_StockIssuedDuringPeriodSharesNewIssues" xlink:label="loc_us-gaapStockIssuedDuringPeriodSharesNewIssues_1270" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapStatementLineItems" xlink:to="loc_us-gaapStockIssuedDuringPeriodSharesNewIssues_1270" xlink:type="arc" order="1" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardDescription" xlink:label="loc_us-gaapShareBasedCompensationArrangementByShareBasedPaymentAwardDescription_1270" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapStatementLineItems" xlink:to="loc_us-gaapShareBasedCompensationArrangementByShareBasedPaymentAwardDescription_1270" xlink:type="arc" order="2" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_StockIssuedDuringPeriodValueStockOptionsExercised" xlink:label="loc_us-gaapStockIssuedDuringPeriodValueStockOptionsExercised_1270" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapStatementLineItems" xlink:to="loc_us-gaapStockIssuedDuringPeriodValueStockOptionsExercised_1270" xlink:type="arc" order="3" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_SharePrice" xlink:label="loc_us-gaapSharePrice_1270" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapStatementLineItems" xlink:to="loc_us-gaapSharePrice_1270" xlink:type="arc" order="4" />
      <link:loc xlink:type="locator" xlink:href="nito-20241231.xsd#NITO_ExercisePriceDescription" xlink:label="loc_NITOExercisePriceDescription_1270" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapStatementLineItems" xlink:to="loc_NITOExercisePriceDescription_1270" xlink:type="arc" order="5" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_SharebasedCompensationArrangementBySharebasedPaymentAwardOptionsVestedInPeriodFairValue1" xlink:label="loc_us-gaapSharebasedCompensationArrangementBySharebasedPaymentAwardOptionsVestedInPeriodFairValue1_1270" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapStatementLineItems" xlink:to="loc_us-gaapSharebasedCompensationArrangementBySharebasedPaymentAwardOptionsVestedInPeriodFairValue1_1270" xlink:type="arc" order="6" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_OfficersCompensation" xlink:label="loc_us-gaapOfficersCompensation_1270" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapStatementLineItems" xlink:to="loc_us-gaapOfficersCompensation_1270" xlink:type="arc" order="7" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_StockIssuedDuringPeriodSharesRestrictedStockAwardGross" xlink:label="loc_us-gaapStockIssuedDuringPeriodSharesRestrictedStockAwardGross_1270" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapStatementLineItems" xlink:to="loc_us-gaapStockIssuedDuringPeriodSharesRestrictedStockAwardGross_1270" xlink:type="arc" order="8" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_StockIssuedDuringPeriodSharesIssuedForServices" xlink:label="loc_us-gaapStockIssuedDuringPeriodSharesIssuedForServices_1270" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapStatementLineItems" xlink:to="loc_us-gaapStockIssuedDuringPeriodSharesIssuedForServices_1270" xlink:type="arc" order="9" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_StockIssuedDuringPeriodValueIssuedForServices" xlink:label="loc_us-gaapStockIssuedDuringPeriodValueIssuedForServices_1270" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapStatementLineItems" xlink:to="loc_us-gaapStockIssuedDuringPeriodValueIssuedForServices_1270" xlink:type="arc" order="10" />
      <link:loc xlink:type="locator" xlink:href="nito-20241231.xsd#NITO_ConsultantFees" xlink:label="loc_NITOConsultantFees_1270" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapStatementLineItems" xlink:to="loc_NITOConsultantFees_1270" xlink:type="arc" order="11" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_ShareBasedCompensation" xlink:label="loc_us-gaapShareBasedCompensation_1270" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapStatementLineItems" xlink:to="loc_us-gaapShareBasedCompensation_1270" xlink:type="arc" order="12" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_StockIssuedDuringPeriodSharesOther" xlink:label="loc_us-gaapStockIssuedDuringPeriodSharesOther_1270" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapStatementLineItems" xlink:to="loc_us-gaapStockIssuedDuringPeriodSharesOther_1270" xlink:type="arc" order="13" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_StockIssuedDuringPeriodValueOther" xlink:label="loc_us-gaapStockIssuedDuringPeriodValueOther_1270" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapStatementLineItems" xlink:to="loc_us-gaapStockIssuedDuringPeriodValueOther_1270" xlink:type="arc" order="14" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_AllocatedShareBasedCompensationExpense" xlink:label="loc_us-gaapAllocatedShareBasedCompensationExpense_1270" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapStatementLineItems" xlink:to="loc_us-gaapAllocatedShareBasedCompensationExpense_1270" xlink:type="arc" order="15" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_EmployeeBenefitsAndShareBasedCompensation" xlink:label="loc_us-gaapEmployeeBenefitsAndShareBasedCompensation_1270" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapStatementLineItems" xlink:to="loc_us-gaapEmployeeBenefitsAndShareBasedCompensation_1270" xlink:type="arc" order="16" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_StockIssuedDuringPeriodValueNewIssues" xlink:label="loc_us-gaapStockIssuedDuringPeriodValueNewIssues_1270" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapStatementLineItems" xlink:to="loc_us-gaapStockIssuedDuringPeriodValueNewIssues_1270" xlink:type="arc" order="17" />
      <link:loc xlink:type="locator" xlink:href="nito-20241231.xsd#NITO_ShareBasedCompensationArrangementByShareBasedPaymentAwardPercentageOfStockIssued" xlink:label="loc_NITOShareBasedCompensationArrangementByShareBasedPaymentAwardPercentageOfStockIssued_1270" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapStatementLineItems" xlink:to="loc_NITOShareBasedCompensationArrangementByShareBasedPaymentAwardPercentageOfStockIssued_1270" xlink:type="arc" order="18" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_StockIssuedDuringPeriodSharesRestrictedStockAwardForfeited" xlink:label="loc_us-gaapStockIssuedDuringPeriodSharesRestrictedStockAwardForfeited_1270" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapStatementLineItems" xlink:to="loc_us-gaapStockIssuedDuringPeriodSharesRestrictedStockAwardForfeited_1270" xlink:type="arc" order="19" />
      <link:loc xlink:type="locator" xlink:href="nito-20241231.xsd#NITO_CashFee" xlink:label="loc_NITOCashFee_1270" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapStatementLineItems" xlink:to="loc_NITOCashFee_1270" xlink:type="arc" order="20" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_StockIssuedDuringPeriodSharesShareBasedCompensation" xlink:label="loc_us-gaapStockIssuedDuringPeriodSharesShareBasedCompensation_1270" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapStatementLineItems" xlink:to="loc_us-gaapStockIssuedDuringPeriodSharesShareBasedCompensation_1270" xlink:type="arc" order="21" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_StockIssuedDuringPeriodValueShareBasedCompensation" xlink:label="loc_us-gaapStockIssuedDuringPeriodValueShareBasedCompensation_1270" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapStatementLineItems" xlink:to="loc_us-gaapStockIssuedDuringPeriodValueShareBasedCompensation_1270" xlink:type="arc" order="22" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_StockIssuedDuringPeriodValueRestrictedStockAwardGross" xlink:label="loc_us-gaapStockIssuedDuringPeriodValueRestrictedStockAwardGross_1270" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapStatementLineItems" xlink:to="loc_us-gaapStockIssuedDuringPeriodValueRestrictedStockAwardGross_1270" xlink:type="arc" order="23" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_Cash" xlink:label="loc_us-gaapCash_1270" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapStatementLineItems" xlink:to="loc_us-gaapCash_1270" xlink:type="arc" order="24" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_LegalFees" xlink:label="loc_us-gaapLegalFees_1270" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapStatementLineItems" xlink:to="loc_us-gaapLegalFees_1270" xlink:type="arc" order="25" />
      <link:loc xlink:type="locator" xlink:href="nito-20241231.xsd#NITO_PurchaseAgreementPercentage" xlink:label="loc_NITOPurchaseAgreementPercentage_1270" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapStatementLineItems" xlink:to="loc_NITOPurchaseAgreementPercentage_1270" xlink:type="arc" order="26" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_DebtInstrumentConvertibleThresholdTradingDays" xlink:label="loc_us-gaapDebtInstrumentConvertibleThresholdTradingDays_1270" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapStatementLineItems" xlink:to="loc_us-gaapDebtInstrumentConvertibleThresholdTradingDays_1270" xlink:type="arc" order="27" />
      <link:loc xlink:type="locator" xlink:href="nito-20241231.xsd#NITO_AdvanceNotice" xlink:label="loc_NITOAdvanceNotice_1270" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapStatementLineItems" xlink:to="loc_NITOAdvanceNotice_1270" xlink:type="arc" order="28" />
      <link:loc xlink:type="locator" xlink:href="nito-20241231.xsd#NITO_AdvanceOrAcquiringPaymentDescription" xlink:label="loc_NITOAdvanceOrAcquiringPaymentDescription_1270" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapStatementLineItems" xlink:to="loc_NITOAdvanceOrAcquiringPaymentDescription_1270" xlink:type="arc" order="29" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_LongTermPurchaseCommitmentAmount" xlink:label="loc_us-gaapLongTermPurchaseCommitmentAmount_1270" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapStatementLineItems" xlink:to="loc_us-gaapLongTermPurchaseCommitmentAmount_1270" xlink:type="arc" order="30" />
      <link:loc xlink:type="locator" xlink:href="nito-20241231.xsd#NITO_PurchaseAgreementDescription" xlink:label="loc_NITOPurchaseAgreementDescription_1270" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapStatementLineItems" xlink:to="loc_NITOPurchaseAgreementDescription_1270" xlink:type="arc" order="31" />
      <link:loc xlink:type="locator" xlink:href="nito-20241231.xsd#NITO_AdvancesDiscountPercentage" xlink:label="loc_NITOAdvancesDiscountPercentage_1270" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapStatementLineItems" xlink:to="loc_NITOAdvancesDiscountPercentage_1270" xlink:type="arc" order="32" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_SaleOfStockNumberOfSharesIssuedInTransaction" xlink:label="loc_us-gaapSaleOfStockNumberOfSharesIssuedInTransaction_1270" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapStatementLineItems" xlink:to="loc_us-gaapSaleOfStockNumberOfSharesIssuedInTransaction_1270" xlink:type="arc" order="33" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_SaleOfStockConsiderationReceivedOnTransaction" xlink:label="loc_us-gaapSaleOfStockConsiderationReceivedOnTransaction_1270" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapStatementLineItems" xlink:to="loc_us-gaapSaleOfStockConsiderationReceivedOnTransaction_1270" xlink:type="arc" order="34" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_ProceedsFromSaleOfIntangibleAssets" xlink:label="loc_us-gaapProceedsFromSaleOfIntangibleAssets_1270" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapStatementLineItems" xlink:to="loc_us-gaapProceedsFromSaleOfIntangibleAssets_1270" xlink:type="arc" order="35" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_GeneralAndAdministrativeExpense" xlink:label="loc_us-gaapGeneralAndAdministrativeExpense_1270" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapStatementLineItems" xlink:to="loc_us-gaapGeneralAndAdministrativeExpense_1270" xlink:type="arc" order="36" />
      <link:loc xlink:type="locator" xlink:href="nito-20241231.xsd#NITO_StructuringFee" xlink:label="loc_NITOStructuringFee_1270" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapStatementLineItems" xlink:to="loc_NITOStructuringFee_1270" xlink:type="arc" order="37" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_PrepaidExpenseCurrent" xlink:label="loc_us-gaapPrepaidExpenseCurrent_1270" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapStatementLineItems" xlink:to="loc_us-gaapPrepaidExpenseCurrent_1270" xlink:type="arc" order="38" />
      <link:loc xlink:type="locator" xlink:href="nito-20241231.xsd#NITO_FinancingExpenses" xlink:label="loc_NITOFinancingExpenses_1270" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapStatementLineItems" xlink:to="loc_NITOFinancingExpenses_1270" xlink:type="arc" order="39" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_PrepaidExpenseCurrentAndNoncurrent" xlink:label="loc_us-gaapPrepaidExpenseCurrentAndNoncurrent_1270" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapStatementLineItems" xlink:to="loc_us-gaapPrepaidExpenseCurrentAndNoncurrent_1270" xlink:type="arc" order="40" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_NotesPayable" xlink:label="loc_us-gaapNotesPayable_1270" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapStatementLineItems" xlink:to="loc_us-gaapNotesPayable_1270" xlink:type="arc" order="41" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_ProceedsFromIssuanceOfDebt" xlink:label="loc_us-gaapProceedsFromIssuanceOfDebt_1270" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapStatementLineItems" xlink:to="loc_us-gaapProceedsFromIssuanceOfDebt_1270" xlink:type="arc" order="42" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_DebtInstrumentInterestRateEffectivePercentage" xlink:label="loc_us-gaapDebtInstrumentInterestRateEffectivePercentage_1270" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapStatementLineItems" xlink:to="loc_us-gaapDebtInstrumentInterestRateEffectivePercentage_1270" xlink:type="arc" order="43" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_DebtInstrumentInterestRateStatedPercentage" xlink:label="loc_us-gaapDebtInstrumentInterestRateStatedPercentage_1270" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapStatementLineItems" xlink:to="loc_us-gaapDebtInstrumentInterestRateStatedPercentage_1270" xlink:type="arc" order="44" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_DebtInstrumentMaturityDate" xlink:label="loc_us-gaapDebtInstrumentMaturityDate_1270" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapStatementLineItems" xlink:to="loc_us-gaapDebtInstrumentMaturityDate_1270" xlink:type="arc" order="45" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_DebtInstrumentPeriodicPayment" xlink:label="loc_us-gaapDebtInstrumentPeriodicPayment_1270" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapStatementLineItems" xlink:to="loc_us-gaapDebtInstrumentPeriodicPayment_1270" xlink:type="arc" order="46" />
      <link:loc xlink:type="locator" xlink:href="nito-20241231.xsd#NITO_DebtInstrumentDefaultInterestRatePercentage" xlink:label="loc_NITODebtInstrumentDefaultInterestRatePercentage_1270" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapStatementLineItems" xlink:to="loc_NITODebtInstrumentDefaultInterestRatePercentage_1270" xlink:type="arc" order="47" />
      <link:loc xlink:type="locator" xlink:href="nito-20241231.xsd#NITO_ProceedsFromStandbyEquityPurchaseAgreement" xlink:label="loc_NITOProceedsFromStandbyEquityPurchaseAgreement_1270" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapStatementLineItems" xlink:to="loc_NITOProceedsFromStandbyEquityPurchaseAgreement_1270" xlink:type="arc" order="48" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_ResearchAndDevelopmentExpense" xlink:label="loc_us-gaapResearchAndDevelopmentExpense_1270" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapStatementLineItems" xlink:to="loc_us-gaapResearchAndDevelopmentExpense_1270" xlink:type="arc" order="49" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_CommonStockParOrStatedValuePerShare" xlink:label="loc_us-gaapCommonStockParOrStatedValuePerShare_1270" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapStatementLineItems" xlink:to="loc_us-gaapCommonStockParOrStatedValuePerShare_1270" xlink:type="arc" order="50" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_ProceedsFromIssuanceOfPrivatePlacement" xlink:label="loc_us-gaapProceedsFromIssuanceOfPrivatePlacement_1270" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapStatementLineItems" xlink:to="loc_us-gaapProceedsFromIssuanceOfPrivatePlacement_1270" xlink:type="arc" order="51" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_SharesIssuedPricePerShare" xlink:label="loc_us-gaapSharesIssuedPricePerShare_1270" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapStatementLineItems" xlink:to="loc_us-gaapSharesIssuedPricePerShare_1270" xlink:type="arc" order="52" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_WarrantsAndRightsOutstandingTerm" xlink:label="loc_us-gaapWarrantsAndRightsOutstandingTerm_1270" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapStatementLineItems" xlink:to="loc_us-gaapWarrantsAndRightsOutstandingTerm_1270" xlink:type="arc" order="53" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_ClassOfWarrantOrRightExercisePriceOfWarrantsOrRights1" xlink:label="loc_us-gaapClassOfWarrantOrRightExercisePriceOfWarrantsOrRights1_1270" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapStatementLineItems" xlink:to="loc_us-gaapClassOfWarrantOrRightExercisePriceOfWarrantsOrRights1_1270" xlink:type="arc" order="54" />
    </link:definitionLink>
    <link:definitionLink xlink:type="extended" xlink:role="http://n2off.com/role/ScheduleOfEstimatedFairValueOfOptionsGrantedDetails" xlink:title="999081 - Disclosure - SCHEDULE OF ESTIMATED FAIR VALUE OF OPTIONS GRANTED (Details)" />
    <link:definitionLink xlink:type="extended" xlink:role="http://n2off.com/role/ScheduleOfEstimatedFairValueOfOptionsGrantedDetailsParenthetical" xlink:title="999082 - Disclosure - SCHEDULE OF ESTIMATED FAIR VALUE OF OPTIONS GRANTED (Details) (Parenthetical)">
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_ShortTermDebtLineItems" xlink:label="loc_us-gaapShortTermDebtLineItems" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_ScheduleOfShortTermDebtTable" xlink:label="loc_us-gaapScheduleOfShortTermDebtTable" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xbrldt:contextElement="segment" xlink:from="loc_us-gaapShortTermDebtLineItems" xlink:to="loc_us-gaapScheduleOfShortTermDebtTable" xlink:type="arc" order="10" xbrldt:closed="true" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_DebtInstrumentAxis" xlink:label="loc_us-gaapDebtInstrumentAxis" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_us-gaapScheduleOfShortTermDebtTable" xlink:to="loc_us-gaapDebtInstrumentAxis" xlink:type="arc" order="1" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_DebtInstrumentNameDomain" xlink:label="loc_us-gaapDebtInstrumentNameDomain" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_us-gaapDebtInstrumentAxis" xlink:to="loc_us-gaapDebtInstrumentNameDomain" xlink:type="arc" order="20" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_DebtInstrumentNameDomain" xlink:label="loc_us-gaapDebtInstrumentNameDomain_10" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xbrldt:contextElement="segment" xlink:from="loc_us-gaapDebtInstrumentAxis" xlink:to="loc_us-gaapDebtInstrumentNameDomain_10" xlink:type="arc" order="30" />
      <link:loc xlink:type="locator" xlink:href="nito-20241231.xsd#NITO_USGovernmentLoansMember" xlink:label="loc_NITOUSGovernmentLoansMember_0" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapDebtInstrumentNameDomain" xlink:to="loc_NITOUSGovernmentLoansMember_0" xlink:type="arc" order="1" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardFairValueAssumptionsRiskFreeInterestRate" xlink:label="loc_us-gaapShareBasedCompensationArrangementByShareBasedPaymentAwardFairValueAssumptionsRiskFreeInterestRate_20" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapShortTermDebtLineItems" xlink:to="loc_us-gaapShareBasedCompensationArrangementByShareBasedPaymentAwardFairValueAssumptionsRiskFreeInterestRate_20" xlink:type="arc" order="0" />
    </link:definitionLink>
    <link:definitionLink xlink:type="extended" xlink:role="http://n2off.com/role/ScheduleOfStockOptionActivityDetails" xlink:title="999083 - Disclosure - SCHEDULE OF STOCK OPTION ACTIVITY (Details)" />
    <link:definitionLink xlink:type="extended" xlink:role="http://n2off.com/role/StockOptionsDetailsNarrative" xlink:title="999084 - Disclosure - STOCK OPTIONS (Details Narrative)">
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems" xlink:label="loc_us-gaapShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_ScheduleOfShareBasedCompensationArrangementsByShareBasedPaymentAwardTable" xlink:label="loc_us-gaapScheduleOfShareBasedCompensationArrangementsByShareBasedPaymentAwardTable" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xbrldt:contextElement="segment" xlink:from="loc_us-gaapShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems" xlink:to="loc_us-gaapScheduleOfShareBasedCompensationArrangementsByShareBasedPaymentAwardTable" xlink:type="arc" order="10" xbrldt:closed="true" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_PlanNameAxis" xlink:label="loc_us-gaapPlanNameAxis" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_us-gaapScheduleOfShareBasedCompensationArrangementsByShareBasedPaymentAwardTable" xlink:to="loc_us-gaapPlanNameAxis" xlink:type="arc" order="1" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_PlanNameDomain" xlink:label="loc_us-gaapPlanNameDomain" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_us-gaapPlanNameAxis" xlink:to="loc_us-gaapPlanNameDomain" xlink:type="arc" order="20" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_PlanNameDomain" xlink:label="loc_us-gaapPlanNameDomain_10" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xbrldt:contextElement="segment" xlink:from="loc_us-gaapPlanNameAxis" xlink:to="loc_us-gaapPlanNameDomain_10" xlink:type="arc" order="30" />
      <link:loc xlink:type="locator" xlink:href="nito-20241231.xsd#NITO_TwoThousandEighteenEquityIncentivePlanMember" xlink:label="loc_NITOTwoThousandEighteenEquityIncentivePlanMember_0" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapPlanNameDomain" xlink:to="loc_NITOTwoThousandEighteenEquityIncentivePlanMember_0" xlink:type="arc" order="1" />
      <link:loc xlink:type="locator" xlink:href="nito-20241231.xsd#NITO_TwoThousandTwentyTwoEquityIncentivePlanMember" xlink:label="loc_NITOTwoThousandTwentyTwoEquityIncentivePlanMember_20" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapPlanNameDomain" xlink:to="loc_NITOTwoThousandTwentyTwoEquityIncentivePlanMember_20" xlink:type="arc" order="21" />
      <link:loc xlink:type="locator" xlink:href="nito-20241231.xsd#NITO_TwoThousandTwentyTwoShareIncentivePlanMember" xlink:label="loc_NITOTwoThousandTwentyTwoShareIncentivePlanMember_40" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapPlanNameDomain" xlink:to="loc_NITOTwoThousandTwentyTwoShareIncentivePlanMember_40" xlink:type="arc" order="41" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/srt/2024/elts/srt-2024.xsd#srt_RangeAxis" xlink:label="loc_srtRangeAxis" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_us-gaapScheduleOfShareBasedCompensationArrangementsByShareBasedPaymentAwardTable" xlink:to="loc_srtRangeAxis" xlink:type="arc" order="2" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/srt/2024/elts/srt-2024.xsd#srt_RangeMember" xlink:label="loc_srtRangeMember" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_srtRangeAxis" xlink:to="loc_srtRangeMember" xlink:type="arc" order="20" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/srt/2024/elts/srt-2024.xsd#srt_RangeMember" xlink:label="loc_srtRangeMember_10" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xbrldt:contextElement="segment" xlink:from="loc_srtRangeAxis" xlink:to="loc_srtRangeMember_10" xlink:type="arc" order="30" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/srt/2024/elts/srt-2024.xsd#srt_MaximumMember" xlink:label="loc_srtMaximumMember_20" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_srtRangeMember" xlink:to="loc_srtMaximumMember_20" xlink:type="arc" order="22" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/srt/2024/elts/srt-2024.xsd#srt_TitleOfIndividualAxis" xlink:label="loc_srtTitleOfIndividualAxis" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_us-gaapScheduleOfShareBasedCompensationArrangementsByShareBasedPaymentAwardTable" xlink:to="loc_srtTitleOfIndividualAxis" xlink:type="arc" order="3" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/srt/2024/elts/srt-2024.xsd#srt_TitleOfIndividualWithRelationshipToEntityDomain" xlink:label="loc_srtTitleOfIndividualWithRelationshipToEntityDomain" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_srtTitleOfIndividualAxis" xlink:to="loc_srtTitleOfIndividualWithRelationshipToEntityDomain" xlink:type="arc" order="20" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/srt/2024/elts/srt-2024.xsd#srt_TitleOfIndividualWithRelationshipToEntityDomain" xlink:label="loc_srtTitleOfIndividualWithRelationshipToEntityDomain_10" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xbrldt:contextElement="segment" xlink:from="loc_srtTitleOfIndividualAxis" xlink:to="loc_srtTitleOfIndividualWithRelationshipToEntityDomain_10" xlink:type="arc" order="30" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/srt/2024/elts/srt-2024.xsd#srt_BoardOfDirectorsChairmanMember" xlink:label="loc_srtBoardOfDirectorsChairmanMember_30" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_srtTitleOfIndividualWithRelationshipToEntityDomain" xlink:to="loc_srtBoardOfDirectorsChairmanMember_30" xlink:type="arc" order="31" />
      <link:loc xlink:type="locator" xlink:href="nito-20241231.xsd#NITO_EmployeesAndDirectorsMember" xlink:label="loc_NITOEmployeesAndDirectorsMember_130" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_srtTitleOfIndividualWithRelationshipToEntityDomain" xlink:to="loc_NITOEmployeesAndDirectorsMember_130" xlink:type="arc" order="131" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_StatementEquityComponentsAxis" xlink:label="loc_us-gaapStatementEquityComponentsAxis" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_us-gaapScheduleOfShareBasedCompensationArrangementsByShareBasedPaymentAwardTable" xlink:to="loc_us-gaapStatementEquityComponentsAxis" xlink:type="arc" order="4" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_EquityComponentDomain" xlink:label="loc_us-gaapEquityComponentDomain" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_us-gaapStatementEquityComponentsAxis" xlink:to="loc_us-gaapEquityComponentDomain" xlink:type="arc" order="20" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_EquityComponentDomain" xlink:label="loc_us-gaapEquityComponentDomain_10" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xbrldt:contextElement="segment" xlink:from="loc_us-gaapStatementEquityComponentsAxis" xlink:to="loc_us-gaapEquityComponentDomain_10" xlink:type="arc" order="30" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_CommonStockMember" xlink:label="loc_us-gaapCommonStockMember_40" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapEquityComponentDomain" xlink:to="loc_us-gaapCommonStockMember_40" xlink:type="arc" order="42" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsExercisableNumber" xlink:label="loc_us-gaapShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsExercisableNumber_180" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems" xlink:to="loc_us-gaapShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsExercisableNumber_180" xlink:type="arc" order="0" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardNumberOfAdditionalSharesAuthorized" xlink:label="loc_us-gaapShareBasedCompensationArrangementByShareBasedPaymentAwardNumberOfAdditionalSharesAuthorized_180" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems" xlink:to="loc_us-gaapShareBasedCompensationArrangementByShareBasedPaymentAwardNumberOfAdditionalSharesAuthorized_180" xlink:type="arc" order="1" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_StockIssuedDuringPeriodSharesEmployeeStockPurchasePlans" xlink:label="loc_us-gaapStockIssuedDuringPeriodSharesEmployeeStockPurchasePlans_180" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems" xlink:to="loc_us-gaapStockIssuedDuringPeriodSharesEmployeeStockPurchasePlans_180" xlink:type="arc" order="2" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsGrantsInPeriodGross" xlink:label="loc_us-gaapShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsGrantsInPeriodGross_180" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems" xlink:to="loc_us-gaapShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsGrantsInPeriodGross_180" xlink:type="arc" order="3" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_StockIssuedDuringPeriodSharesRestrictedStockAwardGross" xlink:label="loc_us-gaapStockIssuedDuringPeriodSharesRestrictedStockAwardGross_180" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems" xlink:to="loc_us-gaapStockIssuedDuringPeriodSharesRestrictedStockAwardGross_180" xlink:type="arc" order="4" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardSharesPurchasedForAward" xlink:label="loc_us-gaapShareBasedCompensationArrangementByShareBasedPaymentAwardSharesPurchasedForAward_180" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems" xlink:to="loc_us-gaapShareBasedCompensationArrangementByShareBasedPaymentAwardSharesPurchasedForAward_180" xlink:type="arc" order="5" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_CommonStockSharesAuthorized" xlink:label="loc_us-gaapCommonStockSharesAuthorized_180" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems" xlink:to="loc_us-gaapCommonStockSharesAuthorized_180" xlink:type="arc" order="6" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_StockIssuedDuringPeriodSharesNewIssues" xlink:label="loc_us-gaapStockIssuedDuringPeriodSharesNewIssues_180" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems" xlink:to="loc_us-gaapStockIssuedDuringPeriodSharesNewIssues_180" xlink:type="arc" order="7" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_SharePrice" xlink:label="loc_us-gaapSharePrice_180" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems" xlink:to="loc_us-gaapSharePrice_180" xlink:type="arc" order="8" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsOutstandingIntrinsicValue" xlink:label="loc_us-gaapShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsOutstandingIntrinsicValue_180" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems" xlink:to="loc_us-gaapShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsOutstandingIntrinsicValue_180" xlink:type="arc" order="9" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_SharesIssuedPricePerShare" xlink:label="loc_us-gaapSharesIssuedPricePerShare_180" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems" xlink:to="loc_us-gaapSharesIssuedPricePerShare_180" xlink:type="arc" order="10" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_EmployeeServiceShareBasedCompensationNonvestedAwardsTotalCompensationCostNotYetRecognizedStockOptions" xlink:label="loc_us-gaapEmployeeServiceShareBasedCompensationNonvestedAwardsTotalCompensationCostNotYetRecognizedStockOptions_180" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems" xlink:to="loc_us-gaapEmployeeServiceShareBasedCompensationNonvestedAwardsTotalCompensationCostNotYetRecognizedStockOptions_180" xlink:type="arc" order="11" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_ShareBasedCompensation" xlink:label="loc_us-gaapShareBasedCompensation_180" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems" xlink:to="loc_us-gaapShareBasedCompensation_180" xlink:type="arc" order="12" />
    </link:definitionLink>
    <link:definitionLink xlink:type="extended" xlink:role="http://n2off.com/role/ScheduleOfCostOfSalesDetails" xlink:title="999085 - Disclosure - SCHEDULE OF COST OF SALES (Details)">
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_StatementLineItems" xlink:label="loc_us-gaapStatementLineItems" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_StatementTable" xlink:label="loc_us-gaapStatementTable" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xbrldt:contextElement="segment" xlink:from="loc_us-gaapStatementLineItems" xlink:to="loc_us-gaapStatementTable" xlink:type="arc" order="10" xbrldt:closed="true" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/srt/2024/elts/srt-2024.xsd#srt_ProductOrServiceAxis" xlink:label="loc_srtProductOrServiceAxis" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_us-gaapStatementTable" xlink:to="loc_srtProductOrServiceAxis" xlink:type="arc" order="1" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/srt/2024/elts/srt-2024.xsd#srt_ProductsAndServicesDomain" xlink:label="loc_srtProductsAndServicesDomain" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_srtProductOrServiceAxis" xlink:to="loc_srtProductsAndServicesDomain" xlink:type="arc" order="20" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/srt/2024/elts/srt-2024.xsd#srt_ProductsAndServicesDomain" xlink:label="loc_srtProductsAndServicesDomain_10" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xbrldt:contextElement="segment" xlink:from="loc_srtProductOrServiceAxis" xlink:to="loc_srtProductsAndServicesDomain_10" xlink:type="arc" order="30" />
      <link:loc xlink:type="locator" xlink:href="nito-20241231.xsd#NITO_SalariesAndRelatedExpensesMember" xlink:label="loc_NITOSalariesAndRelatedExpensesMember_0" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_srtProductsAndServicesDomain" xlink:to="loc_NITOSalariesAndRelatedExpensesMember_0" xlink:type="arc" order="1" />
      <link:loc xlink:type="locator" xlink:href="nito-20241231.xsd#NITO_MaterialsMember" xlink:label="loc_NITOMaterialsMember_20" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_srtProductsAndServicesDomain" xlink:to="loc_NITOMaterialsMember_20" xlink:type="arc" order="21" />
      <link:loc xlink:type="locator" xlink:href="nito-20241231.xsd#NITO_VehicleMaintenanceMember" xlink:label="loc_NITOVehicleMaintenanceMember_40" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_srtProductsAndServicesDomain" xlink:to="loc_NITOVehicleMaintenanceMember_40" xlink:type="arc" order="41" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_IncomeStatementLocationAxis" xlink:label="loc_us-gaapIncomeStatementLocationAxis" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_us-gaapStatementTable" xlink:to="loc_us-gaapIncomeStatementLocationAxis" xlink:type="arc" order="2" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_IncomeStatementLocationDomain" xlink:label="loc_us-gaapIncomeStatementLocationDomain" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_us-gaapIncomeStatementLocationAxis" xlink:to="loc_us-gaapIncomeStatementLocationDomain" xlink:type="arc" order="20" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_IncomeStatementLocationDomain" xlink:label="loc_us-gaapIncomeStatementLocationDomain_10" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xbrldt:contextElement="segment" xlink:from="loc_us-gaapIncomeStatementLocationAxis" xlink:to="loc_us-gaapIncomeStatementLocationDomain_10" xlink:type="arc" order="30" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_OtherExpenseMember" xlink:label="loc_us-gaapOtherExpenseMember_60" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapIncomeStatementLocationDomain" xlink:to="loc_us-gaapOtherExpenseMember_60" xlink:type="arc" order="61" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_CostOfGoodsAndServicesSold" xlink:label="loc_us-gaapCostOfGoodsAndServicesSold_110" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapStatementLineItems" xlink:to="loc_us-gaapCostOfGoodsAndServicesSold_110" xlink:type="arc" order="0" />
    </link:definitionLink>
    <link:definitionLink xlink:type="extended" xlink:role="http://n2off.com/role/ScheduleOfResearchAndDevelopmentExpensesDetails" xlink:title="999086 - Disclosure - SCHEDULE OF RESEARCH AND DEVELOPMENT EXPENSES (Details)">
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_StatementLineItems" xlink:label="loc_us-gaapStatementLineItems" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_StatementTable" xlink:label="loc_us-gaapStatementTable" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xbrldt:contextElement="segment" xlink:from="loc_us-gaapStatementLineItems" xlink:to="loc_us-gaapStatementTable" xlink:type="arc" order="10" xbrldt:closed="true" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/srt/2024/elts/srt-2024.xsd#srt_ProductOrServiceAxis" xlink:label="loc_srtProductOrServiceAxis" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_us-gaapStatementTable" xlink:to="loc_srtProductOrServiceAxis" xlink:type="arc" order="1" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/srt/2024/elts/srt-2024.xsd#srt_ProductsAndServicesDomain" xlink:label="loc_srtProductsAndServicesDomain" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_srtProductOrServiceAxis" xlink:to="loc_srtProductsAndServicesDomain" xlink:type="arc" order="20" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/srt/2024/elts/srt-2024.xsd#srt_ProductsAndServicesDomain" xlink:label="loc_srtProductsAndServicesDomain_10" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xbrldt:contextElement="segment" xlink:from="loc_srtProductOrServiceAxis" xlink:to="loc_srtProductsAndServicesDomain_10" xlink:type="arc" order="30" />
      <link:loc xlink:type="locator" xlink:href="nito-20241231.xsd#NITO_SalariesAndRelatedExpensesMember" xlink:label="loc_NITOSalariesAndRelatedExpensesMember_0" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_srtProductsAndServicesDomain" xlink:to="loc_NITOSalariesAndRelatedExpensesMember_0" xlink:type="arc" order="1" />
      <link:loc xlink:type="locator" xlink:href="nito-20241231.xsd#NITO_IPRAndDMember" xlink:label="loc_NITOIPRAndDMember_20" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_srtProductsAndServicesDomain" xlink:to="loc_NITOIPRAndDMember_20" xlink:type="arc" order="21" />
      <link:loc xlink:type="locator" xlink:href="nito-20241231.xsd#NITO_SubcontractorsMember" xlink:label="loc_NITOSubcontractorsMember_40" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_srtProductsAndServicesDomain" xlink:to="loc_NITOSubcontractorsMember_40" xlink:type="arc" order="41" />
      <link:loc xlink:type="locator" xlink:href="nito-20241231.xsd#NITO_DepreciationMember" xlink:label="loc_NITODepreciationMember_60" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_srtProductsAndServicesDomain" xlink:to="loc_NITODepreciationMember_60" xlink:type="arc" order="61" />
      <link:loc xlink:type="locator" xlink:href="nito-20241231.xsd#NITO_VehicleMaintenanceMember" xlink:label="loc_NITOVehicleMaintenanceMember_80" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_srtProductsAndServicesDomain" xlink:to="loc_NITOVehicleMaintenanceMember_80" xlink:type="arc" order="81" />
      <link:loc xlink:type="locator" xlink:href="nito-20241231.xsd#NITO_LaboratoryAndFieldTestsMember" xlink:label="loc_NITOLaboratoryAndFieldTestsMember_120" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_srtProductsAndServicesDomain" xlink:to="loc_NITOLaboratoryAndFieldTestsMember_120" xlink:type="arc" order="121" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_RelatedPartyTransactionAxis" xlink:label="loc_us-gaapRelatedPartyTransactionAxis" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_us-gaapStatementTable" xlink:to="loc_us-gaapRelatedPartyTransactionAxis" xlink:type="arc" order="2" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_RelatedPartyTransactionDomain" xlink:label="loc_us-gaapRelatedPartyTransactionDomain" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_us-gaapRelatedPartyTransactionAxis" xlink:to="loc_us-gaapRelatedPartyTransactionDomain" xlink:type="arc" order="20" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_RelatedPartyTransactionDomain" xlink:label="loc_us-gaapRelatedPartyTransactionDomain_10" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xbrldt:contextElement="segment" xlink:from="loc_us-gaapRelatedPartyTransactionAxis" xlink:to="loc_us-gaapRelatedPartyTransactionDomain_10" xlink:type="arc" order="30" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_AssetManagementArrangementMember" xlink:label="loc_us-gaapAssetManagementArrangementMember_100" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapRelatedPartyTransactionDomain" xlink:to="loc_us-gaapAssetManagementArrangementMember_100" xlink:type="arc" order="101" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_IncomeStatementLocationAxis" xlink:label="loc_us-gaapIncomeStatementLocationAxis" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_us-gaapStatementTable" xlink:to="loc_us-gaapIncomeStatementLocationAxis" xlink:type="arc" order="3" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_IncomeStatementLocationDomain" xlink:label="loc_us-gaapIncomeStatementLocationDomain" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_us-gaapIncomeStatementLocationAxis" xlink:to="loc_us-gaapIncomeStatementLocationDomain" xlink:type="arc" order="20" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_IncomeStatementLocationDomain" xlink:label="loc_us-gaapIncomeStatementLocationDomain_10" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xbrldt:contextElement="segment" xlink:from="loc_us-gaapIncomeStatementLocationAxis" xlink:to="loc_us-gaapIncomeStatementLocationDomain_10" xlink:type="arc" order="30" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_OtherExpenseMember" xlink:label="loc_us-gaapOtherExpenseMember_140" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapIncomeStatementLocationDomain" xlink:to="loc_us-gaapOtherExpenseMember_140" xlink:type="arc" order="141" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_ResearchAndDevelopmentExpense" xlink:label="loc_us-gaapResearchAndDevelopmentExpense_190" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapStatementLineItems" xlink:to="loc_us-gaapResearchAndDevelopmentExpense_190" xlink:type="arc" order="0" />
    </link:definitionLink>
    <link:definitionLink xlink:type="extended" xlink:role="http://n2off.com/role/ScheduleOfSellingAndMarketingExpensesDetails" xlink:title="999087 - Disclosure - SCHEDULE OF SELLING AND MARKETING EXPENSES (Details)">
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_StatementLineItems" xlink:label="loc_us-gaapStatementLineItems" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_StatementTable" xlink:label="loc_us-gaapStatementTable" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xbrldt:contextElement="segment" xlink:from="loc_us-gaapStatementLineItems" xlink:to="loc_us-gaapStatementTable" xlink:type="arc" order="10" xbrldt:closed="true" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/srt/2024/elts/srt-2024.xsd#srt_ProductOrServiceAxis" xlink:label="loc_srtProductOrServiceAxis" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_us-gaapStatementTable" xlink:to="loc_srtProductOrServiceAxis" xlink:type="arc" order="1" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/srt/2024/elts/srt-2024.xsd#srt_ProductsAndServicesDomain" xlink:label="loc_srtProductsAndServicesDomain" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_srtProductOrServiceAxis" xlink:to="loc_srtProductsAndServicesDomain" xlink:type="arc" order="20" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/srt/2024/elts/srt-2024.xsd#srt_ProductsAndServicesDomain" xlink:label="loc_srtProductsAndServicesDomain_10" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xbrldt:contextElement="segment" xlink:from="loc_srtProductOrServiceAxis" xlink:to="loc_srtProductsAndServicesDomain_10" xlink:type="arc" order="30" />
      <link:loc xlink:type="locator" xlink:href="nito-20241231.xsd#NITO_SalariesAndRelatedExpensesMember" xlink:label="loc_NITOSalariesAndRelatedExpensesMember_0" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_srtProductsAndServicesDomain" xlink:to="loc_NITOSalariesAndRelatedExpensesMember_0" xlink:type="arc" order="1" />
      <link:loc xlink:type="locator" xlink:href="nito-20241231.xsd#NITO_SubcontractorsMember" xlink:label="loc_NITOSubcontractorsMember_40" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_srtProductsAndServicesDomain" xlink:to="loc_NITOSubcontractorsMember_40" xlink:type="arc" order="41" />
      <link:loc xlink:type="locator" xlink:href="nito-20241231.xsd#NITO_TransportAndStorageMember" xlink:label="loc_NITOTransportAndStorageMember_60" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_srtProductsAndServicesDomain" xlink:to="loc_NITOTransportAndStorageMember_60" xlink:type="arc" order="61" />
      <link:loc xlink:type="locator" xlink:href="nito-20241231.xsd#NITO_CommissionsMember" xlink:label="loc_NITOCommissionsMember_80" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_srtProductsAndServicesDomain" xlink:to="loc_NITOCommissionsMember_80" xlink:type="arc" order="81" />
      <link:loc xlink:type="locator" xlink:href="nito-20241231.xsd#NITO_TravelExpensesMember" xlink:label="loc_NITOTravelExpensesMember_100" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_srtProductsAndServicesDomain" xlink:to="loc_NITOTravelExpensesMember_100" xlink:type="arc" order="101" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_AwardTypeAxis" xlink:label="loc_us-gaapAwardTypeAxis" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_us-gaapStatementTable" xlink:to="loc_us-gaapAwardTypeAxis" xlink:type="arc" order="2" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain" xlink:label="loc_us-gaapShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_us-gaapAwardTypeAxis" xlink:to="loc_us-gaapShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain" xlink:type="arc" order="20" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain" xlink:label="loc_us-gaapShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain_10" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xbrldt:contextElement="segment" xlink:from="loc_us-gaapAwardTypeAxis" xlink:to="loc_us-gaapShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain_10" xlink:type="arc" order="30" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_StockCompensationPlanMember" xlink:label="loc_us-gaapStockCompensationPlanMember_20" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain" xlink:to="loc_us-gaapStockCompensationPlanMember_20" xlink:type="arc" order="21" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_IncomeStatementLocationAxis" xlink:label="loc_us-gaapIncomeStatementLocationAxis" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_us-gaapStatementTable" xlink:to="loc_us-gaapIncomeStatementLocationAxis" xlink:type="arc" order="3" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_IncomeStatementLocationDomain" xlink:label="loc_us-gaapIncomeStatementLocationDomain" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_us-gaapIncomeStatementLocationAxis" xlink:to="loc_us-gaapIncomeStatementLocationDomain" xlink:type="arc" order="20" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_IncomeStatementLocationDomain" xlink:label="loc_us-gaapIncomeStatementLocationDomain_10" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xbrldt:contextElement="segment" xlink:from="loc_us-gaapIncomeStatementLocationAxis" xlink:to="loc_us-gaapIncomeStatementLocationDomain_10" xlink:type="arc" order="30" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_OtherExpenseMember" xlink:label="loc_us-gaapOtherExpenseMember_120" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapIncomeStatementLocationDomain" xlink:to="loc_us-gaapOtherExpenseMember_120" xlink:type="arc" order="121" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_SellingAndMarketingExpense" xlink:label="loc_us-gaapSellingAndMarketingExpense_170" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapStatementLineItems" xlink:to="loc_us-gaapSellingAndMarketingExpense_170" xlink:type="arc" order="0" />
    </link:definitionLink>
    <link:definitionLink xlink:type="extended" xlink:role="http://n2off.com/role/ScheduleOfGeneralAndAdministrativeExpensesDetails" xlink:title="999088 - Disclosure - SCHEDULE OF GENERAL AND ADMINISTRATIVE EXPENSES (Details)">
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_StatementLineItems" xlink:label="loc_us-gaapStatementLineItems" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_StatementTable" xlink:label="loc_us-gaapStatementTable" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xbrldt:contextElement="segment" xlink:from="loc_us-gaapStatementLineItems" xlink:to="loc_us-gaapStatementTable" xlink:type="arc" order="10" xbrldt:closed="true" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/srt/2024/elts/srt-2024.xsd#srt_ProductOrServiceAxis" xlink:label="loc_srtProductOrServiceAxis" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_us-gaapStatementTable" xlink:to="loc_srtProductOrServiceAxis" xlink:type="arc" order="1" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/srt/2024/elts/srt-2024.xsd#srt_ProductsAndServicesDomain" xlink:label="loc_srtProductsAndServicesDomain" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_srtProductOrServiceAxis" xlink:to="loc_srtProductsAndServicesDomain" xlink:type="arc" order="20" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/srt/2024/elts/srt-2024.xsd#srt_ProductsAndServicesDomain" xlink:label="loc_srtProductsAndServicesDomain_10" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xbrldt:contextElement="segment" xlink:from="loc_srtProductOrServiceAxis" xlink:to="loc_srtProductsAndServicesDomain_10" xlink:type="arc" order="30" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_ServiceMember" xlink:label="loc_us-gaapServiceMember_0" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_srtProductsAndServicesDomain" xlink:to="loc_us-gaapServiceMember_0" xlink:type="arc" order="1" />
      <link:loc xlink:type="locator" xlink:href="nito-20241231.xsd#NITO_SalariesAndRelatedExpensesMember" xlink:label="loc_NITOSalariesAndRelatedExpensesMember_20" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_srtProductsAndServicesDomain" xlink:to="loc_NITOSalariesAndRelatedExpensesMember_20" xlink:type="arc" order="21" />
      <link:loc xlink:type="locator" xlink:href="nito-20241231.xsd#NITO_LegalExpensesMember" xlink:label="loc_NITOLegalExpensesMember_60" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_srtProductsAndServicesDomain" xlink:to="loc_NITOLegalExpensesMember_60" xlink:type="arc" order="61" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_MaintenanceMember" xlink:label="loc_us-gaapMaintenanceMember_100" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_srtProductsAndServicesDomain" xlink:to="loc_us-gaapMaintenanceMember_100" xlink:type="arc" order="101" />
      <link:loc xlink:type="locator" xlink:href="nito-20241231.xsd#NITO_RegistrationFeesMember" xlink:label="loc_NITORegistrationFeesMember_120" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_srtProductsAndServicesDomain" xlink:to="loc_NITORegistrationFeesMember_120" xlink:type="arc" order="121" />
      <link:loc xlink:type="locator" xlink:href="nito-20241231.xsd#NITO_DoubtfulDebtMember" xlink:label="loc_NITODoubtfulDebtMember_140" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_srtProductsAndServicesDomain" xlink:to="loc_NITODoubtfulDebtMember_140" xlink:type="arc" order="141" />
      <link:loc xlink:type="locator" xlink:href="nito-20241231.xsd#NITO_DepreciationMember" xlink:label="loc_NITODepreciationMember_160" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_srtProductsAndServicesDomain" xlink:to="loc_NITODepreciationMember_160" xlink:type="arc" order="161" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_AwardTypeAxis" xlink:label="loc_us-gaapAwardTypeAxis" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_us-gaapStatementTable" xlink:to="loc_us-gaapAwardTypeAxis" xlink:type="arc" order="2" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain" xlink:label="loc_us-gaapShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_us-gaapAwardTypeAxis" xlink:to="loc_us-gaapShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain" xlink:type="arc" order="20" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain" xlink:label="loc_us-gaapShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain_10" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xbrldt:contextElement="segment" xlink:from="loc_us-gaapAwardTypeAxis" xlink:to="loc_us-gaapShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain_10" xlink:type="arc" order="30" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_StockCompensationPlanMember" xlink:label="loc_us-gaapStockCompensationPlanMember_40" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain" xlink:to="loc_us-gaapStockCompensationPlanMember_40" xlink:type="arc" order="41" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_LossContingenciesByNatureOfContingencyAxis" xlink:label="loc_us-gaapLossContingenciesByNatureOfContingencyAxis" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_us-gaapStatementTable" xlink:to="loc_us-gaapLossContingenciesByNatureOfContingencyAxis" xlink:type="arc" order="3" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_LossContingencyNatureDomain" xlink:label="loc_us-gaapLossContingencyNatureDomain" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_us-gaapLossContingenciesByNatureOfContingencyAxis" xlink:to="loc_us-gaapLossContingencyNatureDomain" xlink:type="arc" order="20" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_LossContingencyNatureDomain" xlink:label="loc_us-gaapLossContingencyNatureDomain_10" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xbrldt:contextElement="segment" xlink:from="loc_us-gaapLossContingenciesByNatureOfContingencyAxis" xlink:to="loc_us-gaapLossContingencyNatureDomain_10" xlink:type="arc" order="30" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_InsuranceRelatedAssessmentsMember" xlink:label="loc_us-gaapInsuranceRelatedAssessmentsMember_80" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapLossContingencyNatureDomain" xlink:to="loc_us-gaapInsuranceRelatedAssessmentsMember_80" xlink:type="arc" order="81" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_IncomeStatementLocationAxis" xlink:label="loc_us-gaapIncomeStatementLocationAxis" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_us-gaapStatementTable" xlink:to="loc_us-gaapIncomeStatementLocationAxis" xlink:type="arc" order="4" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_IncomeStatementLocationDomain" xlink:label="loc_us-gaapIncomeStatementLocationDomain" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_us-gaapIncomeStatementLocationAxis" xlink:to="loc_us-gaapIncomeStatementLocationDomain" xlink:type="arc" order="20" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_IncomeStatementLocationDomain" xlink:label="loc_us-gaapIncomeStatementLocationDomain_10" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xbrldt:contextElement="segment" xlink:from="loc_us-gaapIncomeStatementLocationAxis" xlink:to="loc_us-gaapIncomeStatementLocationDomain_10" xlink:type="arc" order="30" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_OtherExpenseMember" xlink:label="loc_us-gaapOtherExpenseMember_180" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapIncomeStatementLocationDomain" xlink:to="loc_us-gaapOtherExpenseMember_180" xlink:type="arc" order="181" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_GeneralAndAdministrativeExpense" xlink:label="loc_us-gaapGeneralAndAdministrativeExpense_230" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapStatementLineItems" xlink:to="loc_us-gaapGeneralAndAdministrativeExpense_230" xlink:type="arc" order="0" />
      <link:loc xlink:type="locator" xlink:href="nito-20241231.xsd#NITO_DoubtfulDebt" xlink:label="loc_NITODoubtfulDebt_230" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapStatementLineItems" xlink:to="loc_NITODoubtfulDebt_230" xlink:type="arc" order="1" />
    </link:definitionLink>
    <link:definitionLink xlink:type="extended" xlink:role="http://n2off.com/role/ScheduleOfFinancingExpensesNetDetails" xlink:title="999089 - Disclosure - SCHEDULE OF FINANCING EXPENSES NET (Details)" />
    <link:definitionLink xlink:type="extended" xlink:role="http://n2off.com/role/ScheduleOfIncomeTaxExpensesDetails" xlink:title="999090 - Disclosure - SCHEDULE OF INCOME TAX EXPENSES (Details)" />
    <link:definitionLink xlink:type="extended" xlink:role="http://n2off.com/role/ScheduleOfDeferredTaxAssetsAndLiabilitiesDetails" xlink:title="999091 - Disclosure - SCHEDULE OF DEFERRED TAX ASSETS AND LIABILITIES (Details)" />
    <link:definitionLink xlink:type="extended" xlink:role="http://n2off.com/role/IncomeTaxDetailsNarrative" xlink:title="999092 - Disclosure - INCOME TAX (Details Narrative)">
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_SignificantChangeInUnrecognizedTaxBenefitsIsReasonablyPossibleLineItems" xlink:label="loc_us-gaapSignificantChangeInUnrecognizedTaxBenefitsIsReasonablyPossibleLineItems" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_SignificantChangeInUnrecognizedTaxBenefitsIsReasonablyPossibleTable" xlink:label="loc_us-gaapSignificantChangeInUnrecognizedTaxBenefitsIsReasonablyPossibleTable" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xbrldt:contextElement="segment" xlink:from="loc_us-gaapSignificantChangeInUnrecognizedTaxBenefitsIsReasonablyPossibleLineItems" xlink:to="loc_us-gaapSignificantChangeInUnrecognizedTaxBenefitsIsReasonablyPossibleTable" xlink:type="arc" order="10" xbrldt:closed="true" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/srt/2024/elts/srt-2024.xsd#srt_RangeAxis" xlink:label="loc_srtRangeAxis" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_us-gaapSignificantChangeInUnrecognizedTaxBenefitsIsReasonablyPossibleTable" xlink:to="loc_srtRangeAxis" xlink:type="arc" order="1" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/srt/2024/elts/srt-2024.xsd#srt_RangeMember" xlink:label="loc_srtRangeMember" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_srtRangeAxis" xlink:to="loc_srtRangeMember" xlink:type="arc" order="20" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/srt/2024/elts/srt-2024.xsd#srt_RangeMember" xlink:label="loc_srtRangeMember_10" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xbrldt:contextElement="segment" xlink:from="loc_srtRangeAxis" xlink:to="loc_srtRangeMember_10" xlink:type="arc" order="30" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/srt/2024/elts/srt-2024.xsd#srt_MinimumMember" xlink:label="loc_srtMinimumMember_10" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_srtRangeMember" xlink:to="loc_srtMinimumMember_10" xlink:type="arc" order="11" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/srt/2024/elts/srt-2024.xsd#srt_MaximumMember" xlink:label="loc_srtMaximumMember_40" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_srtRangeMember" xlink:to="loc_srtMaximumMember_40" xlink:type="arc" order="41" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_EffectiveIncomeTaxRateReconciliationAtFederalStatutoryIncomeTaxRate" xlink:label="loc_us-gaapEffectiveIncomeTaxRateReconciliationAtFederalStatutoryIncomeTaxRate_60" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapSignificantChangeInUnrecognizedTaxBenefitsIsReasonablyPossibleLineItems" xlink:to="loc_us-gaapEffectiveIncomeTaxRateReconciliationAtFederalStatutoryIncomeTaxRate_60" xlink:type="arc" order="0" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_IncomeTaxExaminationDescription" xlink:label="loc_us-gaapIncomeTaxExaminationDescription_60" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapSignificantChangeInUnrecognizedTaxBenefitsIsReasonablyPossibleLineItems" xlink:to="loc_us-gaapIncomeTaxExaminationDescription_60" xlink:type="arc" order="1" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_OperatingLossCarryforwards" xlink:label="loc_us-gaapOperatingLossCarryforwards_60" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapSignificantChangeInUnrecognizedTaxBenefitsIsReasonablyPossibleLineItems" xlink:to="loc_us-gaapOperatingLossCarryforwards_60" xlink:type="arc" order="2" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_IncomeLossFromContinuingOperationsBeforeIncomeTaxesDomestic" xlink:label="loc_us-gaapIncomeLossFromContinuingOperationsBeforeIncomeTaxesDomestic_60" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapSignificantChangeInUnrecognizedTaxBenefitsIsReasonablyPossibleLineItems" xlink:to="loc_us-gaapIncomeLossFromContinuingOperationsBeforeIncomeTaxesDomestic_60" xlink:type="arc" order="3" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_ValuationAllowanceDeferredTaxAssetChangeInAmount" xlink:label="loc_us-gaapValuationAllowanceDeferredTaxAssetChangeInAmount_60" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapSignificantChangeInUnrecognizedTaxBenefitsIsReasonablyPossibleLineItems" xlink:to="loc_us-gaapValuationAllowanceDeferredTaxAssetChangeInAmount_60" xlink:type="arc" order="4" />
    </link:definitionLink>
    <link:definitionLink xlink:type="extended" xlink:role="http://n2off.com/role/ScheduleOfBasicAndDilutedLossPerShareDetails" xlink:title="999093 - Disclosure - SCHEDULE OF BASIC AND DILUTED LOSS PER SHARE (Details)" />
    <link:definitionLink xlink:type="extended" xlink:role="http://n2off.com/role/ScheduleOfTransactionsAndBalancesWithRelatedPartiesDetails" xlink:title="999094 - Disclosure - SCHEDULE OF TRANSACTIONS AND BALANCES WITH RELATED PARTIES (Details)">
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_RelatedPartyTransactionLineItems" xlink:label="loc_us-gaapRelatedPartyTransactionLineItems" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_ScheduleOfRelatedPartyTransactionsByRelatedPartyTable" xlink:label="loc_us-gaapScheduleOfRelatedPartyTransactionsByRelatedPartyTable" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xbrldt:contextElement="segment" xlink:from="loc_us-gaapRelatedPartyTransactionLineItems" xlink:to="loc_us-gaapScheduleOfRelatedPartyTransactionsByRelatedPartyTable" xlink:type="arc" order="10" xbrldt:closed="true" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_IncomeStatementLocationAxis" xlink:label="loc_us-gaapIncomeStatementLocationAxis" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_us-gaapScheduleOfRelatedPartyTransactionsByRelatedPartyTable" xlink:to="loc_us-gaapIncomeStatementLocationAxis" xlink:type="arc" order="1" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_IncomeStatementLocationDomain" xlink:label="loc_us-gaapIncomeStatementLocationDomain" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_us-gaapIncomeStatementLocationAxis" xlink:to="loc_us-gaapIncomeStatementLocationDomain" xlink:type="arc" order="20" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_IncomeStatementLocationDomain" xlink:label="loc_us-gaapIncomeStatementLocationDomain_10" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xbrldt:contextElement="segment" xlink:from="loc_us-gaapIncomeStatementLocationAxis" xlink:to="loc_us-gaapIncomeStatementLocationDomain_10" xlink:type="arc" order="30" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_GeneralAndAdministrativeExpenseMember" xlink:label="loc_us-gaapGeneralAndAdministrativeExpenseMember_0" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapIncomeStatementLocationDomain" xlink:to="loc_us-gaapGeneralAndAdministrativeExpenseMember_0" xlink:type="arc" order="1" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_ResearchAndDevelopmentExpenseMember" xlink:label="loc_us-gaapResearchAndDevelopmentExpenseMember_60" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapIncomeStatementLocationDomain" xlink:to="loc_us-gaapResearchAndDevelopmentExpenseMember_60" xlink:type="arc" order="61" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_SellingAndMarketingExpenseMember" xlink:label="loc_us-gaapSellingAndMarketingExpenseMember_80" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapIncomeStatementLocationDomain" xlink:to="loc_us-gaapSellingAndMarketingExpenseMember_80" xlink:type="arc" order="81" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/srt/2024/elts/srt-2024.xsd#srt_TitleOfIndividualAxis" xlink:label="loc_srtTitleOfIndividualAxis" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_us-gaapScheduleOfRelatedPartyTransactionsByRelatedPartyTable" xlink:to="loc_srtTitleOfIndividualAxis" xlink:type="arc" order="2" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/srt/2024/elts/srt-2024.xsd#srt_TitleOfIndividualWithRelationshipToEntityDomain" xlink:label="loc_srtTitleOfIndividualWithRelationshipToEntityDomain" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_srtTitleOfIndividualAxis" xlink:to="loc_srtTitleOfIndividualWithRelationshipToEntityDomain" xlink:type="arc" order="20" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/srt/2024/elts/srt-2024.xsd#srt_TitleOfIndividualWithRelationshipToEntityDomain" xlink:label="loc_srtTitleOfIndividualWithRelationshipToEntityDomain_10" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xbrldt:contextElement="segment" xlink:from="loc_srtTitleOfIndividualAxis" xlink:to="loc_srtTitleOfIndividualWithRelationshipToEntityDomain_10" xlink:type="arc" order="30" />
      <link:loc xlink:type="locator" xlink:href="nito-20241231.xsd#NITO_DirectorsCompensationMember" xlink:label="loc_NITODirectorsCompensationMember_0" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_srtTitleOfIndividualWithRelationshipToEntityDomain" xlink:to="loc_NITODirectorsCompensationMember_0" xlink:type="arc" order="2" />
      <link:loc xlink:type="locator" xlink:href="nito-20241231.xsd#NITO_SalariesAndFeesToOfficersMember" xlink:label="loc_NITOSalariesAndFeesToOfficersMember_20" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_srtTitleOfIndividualWithRelationshipToEntityDomain" xlink:to="loc_NITOSalariesAndFeesToOfficersMember_20" xlink:type="arc" order="22" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_RelatedPartyTransactionAxis" xlink:label="loc_us-gaapRelatedPartyTransactionAxis" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_us-gaapScheduleOfRelatedPartyTransactionsByRelatedPartyTable" xlink:to="loc_us-gaapRelatedPartyTransactionAxis" xlink:type="arc" order="3" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_RelatedPartyTransactionDomain" xlink:label="loc_us-gaapRelatedPartyTransactionDomain" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_us-gaapRelatedPartyTransactionAxis" xlink:to="loc_us-gaapRelatedPartyTransactionDomain" xlink:type="arc" order="20" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_RelatedPartyTransactionDomain" xlink:label="loc_us-gaapRelatedPartyTransactionDomain_10" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xbrldt:contextElement="segment" xlink:from="loc_us-gaapRelatedPartyTransactionAxis" xlink:to="loc_us-gaapRelatedPartyTransactionDomain_10" xlink:type="arc" order="30" />
      <link:loc xlink:type="locator" xlink:href="nito-20241231.xsd#NITO_RelatedPartiesAndOfficersMember" xlink:label="loc_NITORelatedPartiesAndOfficersMember_100" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapRelatedPartyTransactionDomain" xlink:to="loc_NITORelatedPartiesAndOfficersMember_100" xlink:type="arc" order="101" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_GeneralAndAdministrativeExpense" xlink:label="loc_us-gaapGeneralAndAdministrativeExpense_130" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapRelatedPartyTransactionLineItems" xlink:to="loc_us-gaapGeneralAndAdministrativeExpense_130" xlink:type="arc" order="0" />
      <link:loc xlink:type="locator" xlink:href="nito-20241231.xsd#NITO_GeneralAndAdministrativeExpenses" xlink:label="loc_NITOGeneralAndAdministrativeExpenses_130" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapRelatedPartyTransactionLineItems" xlink:to="loc_NITOGeneralAndAdministrativeExpenses_130" xlink:type="arc" order="1" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_ShareBasedCompensation" xlink:label="loc_us-gaapShareBasedCompensation_130" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapRelatedPartyTransactionLineItems" xlink:to="loc_us-gaapShareBasedCompensation_130" xlink:type="arc" order="2" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_ResearchAndDevelopmentExpense" xlink:label="loc_us-gaapResearchAndDevelopmentExpense_130" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapRelatedPartyTransactionLineItems" xlink:to="loc_us-gaapResearchAndDevelopmentExpense_130" xlink:type="arc" order="3" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_SellingAndMarketingExpense" xlink:label="loc_us-gaapSellingAndMarketingExpense_130" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapRelatedPartyTransactionLineItems" xlink:to="loc_us-gaapSellingAndMarketingExpense_130" xlink:type="arc" order="4" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_AccountsPayableOtherCurrentAndNoncurrent" xlink:label="loc_us-gaapAccountsPayableOtherCurrentAndNoncurrent_130" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapRelatedPartyTransactionLineItems" xlink:to="loc_us-gaapAccountsPayableOtherCurrentAndNoncurrent_130" xlink:type="arc" order="5" />
    </link:definitionLink>
    <link:definitionLink xlink:type="extended" xlink:role="http://n2off.com/role/RelatedPartiesDetailsNarrative" xlink:title="999095 - Disclosure - RELATED PARTIES (Details Narrative)">
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_StatementLineItems" xlink:label="loc_us-gaapStatementLineItems" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_StatementTable" xlink:label="loc_us-gaapStatementTable" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xbrldt:contextElement="segment" xlink:from="loc_us-gaapStatementLineItems" xlink:to="loc_us-gaapStatementTable" xlink:type="arc" order="10" xbrldt:closed="true" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/srt/2024/elts/srt-2024.xsd#srt_TitleOfIndividualAxis" xlink:label="loc_srtTitleOfIndividualAxis" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_us-gaapStatementTable" xlink:to="loc_srtTitleOfIndividualAxis" xlink:type="arc" order="1" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/srt/2024/elts/srt-2024.xsd#srt_TitleOfIndividualWithRelationshipToEntityDomain" xlink:label="loc_srtTitleOfIndividualWithRelationshipToEntityDomain" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_srtTitleOfIndividualAxis" xlink:to="loc_srtTitleOfIndividualWithRelationshipToEntityDomain" xlink:type="arc" order="20" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/srt/2024/elts/srt-2024.xsd#srt_TitleOfIndividualWithRelationshipToEntityDomain" xlink:label="loc_srtTitleOfIndividualWithRelationshipToEntityDomain_10" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xbrldt:contextElement="segment" xlink:from="loc_srtTitleOfIndividualAxis" xlink:to="loc_srtTitleOfIndividualWithRelationshipToEntityDomain_10" xlink:type="arc" order="30" />
      <link:loc xlink:type="locator" xlink:href="nito-20241231.xsd#NITO_Mr.DavidPalachMember" xlink:label="loc_NITOMr.DavidPalachMember_0" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_srtTitleOfIndividualWithRelationshipToEntityDomain" xlink:to="loc_NITOMr.DavidPalachMember_0" xlink:type="arc" order="1" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/srt/2024/elts/srt-2024.xsd#srt_BoardOfDirectorsChairmanMember" xlink:label="loc_srtBoardOfDirectorsChairmanMember_60" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_srtTitleOfIndividualWithRelationshipToEntityDomain" xlink:to="loc_srtBoardOfDirectorsChairmanMember_60" xlink:type="arc" order="61" />
      <link:loc xlink:type="locator" xlink:href="nito-20241231.xsd#NITO_MsLitalBardaMember" xlink:label="loc_NITOMsLitalBardaMember_200" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_srtTitleOfIndividualWithRelationshipToEntityDomain" xlink:to="loc_NITOMsLitalBardaMember_200" xlink:type="arc" order="201" />
      <link:loc xlink:type="locator" xlink:href="nito-20241231.xsd#NITO_MsBardaMember" xlink:label="loc_NITOMsBardaMember_210" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_srtTitleOfIndividualWithRelationshipToEntityDomain" xlink:to="loc_NITOMsBardaMember_210" xlink:type="arc" order="211" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_TypeOfArrangementAxis" xlink:label="loc_us-gaapTypeOfArrangementAxis" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_us-gaapStatementTable" xlink:to="loc_us-gaapTypeOfArrangementAxis" xlink:type="arc" order="2" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_ArrangementsAndNonarrangementTransactionsMember" xlink:label="loc_us-gaapArrangementsAndNonarrangementTransactionsMember" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_us-gaapTypeOfArrangementAxis" xlink:to="loc_us-gaapArrangementsAndNonarrangementTransactionsMember" xlink:type="arc" order="20" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_ArrangementsAndNonarrangementTransactionsMember" xlink:label="loc_us-gaapArrangementsAndNonarrangementTransactionsMember_10" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xbrldt:contextElement="segment" xlink:from="loc_us-gaapTypeOfArrangementAxis" xlink:to="loc_us-gaapArrangementsAndNonarrangementTransactionsMember_10" xlink:type="arc" order="30" />
      <link:loc xlink:type="locator" xlink:href="nito-20241231.xsd#NITO_ConsultingAgreementMember" xlink:label="loc_NITOConsultingAgreementMember_0" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapArrangementsAndNonarrangementTransactionsMember" xlink:to="loc_NITOConsultingAgreementMember_0" xlink:type="arc" order="2" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_StatementEquityComponentsAxis" xlink:label="loc_us-gaapStatementEquityComponentsAxis" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_us-gaapStatementTable" xlink:to="loc_us-gaapStatementEquityComponentsAxis" xlink:type="arc" order="3" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_EquityComponentDomain" xlink:label="loc_us-gaapEquityComponentDomain" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_us-gaapStatementEquityComponentsAxis" xlink:to="loc_us-gaapEquityComponentDomain" xlink:type="arc" order="20" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_EquityComponentDomain" xlink:label="loc_us-gaapEquityComponentDomain_10" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xbrldt:contextElement="segment" xlink:from="loc_us-gaapStatementEquityComponentsAxis" xlink:to="loc_us-gaapEquityComponentDomain_10" xlink:type="arc" order="30" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_CommonStockMember" xlink:label="loc_us-gaapCommonStockMember_30" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapEquityComponentDomain" xlink:to="loc_us-gaapCommonStockMember_30" xlink:type="arc" order="32" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/srt/2024/elts/srt-2024.xsd#srt_RangeAxis" xlink:label="loc_srtRangeAxis" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_us-gaapStatementTable" xlink:to="loc_srtRangeAxis" xlink:type="arc" order="4" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/srt/2024/elts/srt-2024.xsd#srt_RangeMember" xlink:label="loc_srtRangeMember" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_srtRangeAxis" xlink:to="loc_srtRangeMember" xlink:type="arc" order="20" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/srt/2024/elts/srt-2024.xsd#srt_RangeMember" xlink:label="loc_srtRangeMember_10" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xbrldt:contextElement="segment" xlink:from="loc_srtRangeAxis" xlink:to="loc_srtRangeMember_10" xlink:type="arc" order="30" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/srt/2024/elts/srt-2024.xsd#srt_MaximumMember" xlink:label="loc_srtMaximumMember_50" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_srtRangeMember" xlink:to="loc_srtMaximumMember_50" xlink:type="arc" order="51" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/srt/2024/elts/srt-2024.xsd#srt_MinimumMember" xlink:label="loc_srtMinimumMember_90" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_srtRangeMember" xlink:to="loc_srtMinimumMember_90" xlink:type="arc" order="92" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_SalariesWagesAndOfficersCompensation" xlink:label="loc_us-gaapSalariesWagesAndOfficersCompensation_230" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapStatementLineItems" xlink:to="loc_us-gaapSalariesWagesAndOfficersCompensation_230" xlink:type="arc" order="0" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_DebtInstrumentPeriodicPayment" xlink:label="loc_us-gaapDebtInstrumentPeriodicPayment_230" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapStatementLineItems" xlink:to="loc_us-gaapDebtInstrumentPeriodicPayment_230" xlink:type="arc" order="1" />
      <link:loc xlink:type="locator" xlink:href="nito-20241231.xsd#NITO_ReimbursementExpenses" xlink:label="loc_NITOReimbursementExpenses_230" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapStatementLineItems" xlink:to="loc_NITOReimbursementExpenses_230" xlink:type="arc" order="2" />
      <link:loc xlink:type="locator" xlink:href="nito-20241231.xsd#NITO_CommonStockOutstandingPercentage" xlink:label="loc_NITOCommonStockOutstandingPercentage_230" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapStatementLineItems" xlink:to="loc_NITOCommonStockOutstandingPercentage_230" xlink:type="arc" order="3" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_NotesPayable" xlink:label="loc_us-gaapNotesPayable_230" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapStatementLineItems" xlink:to="loc_us-gaapNotesPayable_230" xlink:type="arc" order="4" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_StockIssuedDuringPeriodSharesNewIssues" xlink:label="loc_us-gaapStockIssuedDuringPeriodSharesNewIssues_230" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapStatementLineItems" xlink:to="loc_us-gaapStockIssuedDuringPeriodSharesNewIssues_230" xlink:type="arc" order="5" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_SalariesAndWages" xlink:label="loc_us-gaapSalariesAndWages_230" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapStatementLineItems" xlink:to="loc_us-gaapSalariesAndWages_230" xlink:type="arc" order="6" />
      <link:loc xlink:type="locator" xlink:href="nito-20241231.xsd#NITO_AnnualFees" xlink:label="loc_NITOAnnualFees_230" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapStatementLineItems" xlink:to="loc_NITOAnnualFees_230" xlink:type="arc" order="7" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_DeferredCompensationArrangementsOverallDescription" xlink:label="loc_us-gaapDeferredCompensationArrangementsOverallDescription_230" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapStatementLineItems" xlink:to="loc_us-gaapDeferredCompensationArrangementsOverallDescription_230" xlink:type="arc" order="8" />
    </link:definitionLink>
    <link:definitionLink xlink:type="extended" xlink:role="http://n2off.com/role/ScheduleOfInformationRelatedToOperationsOfReportableOperatingSegmentsDetails" xlink:title="999096 - Disclosure - SCHEDULE OF INFORMATION RELATED TO OPERATIONS OF REPORTABLE OPERATING SEGMENTS (Details)">
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_SegmentReportingInformationLineItems" xlink:label="loc_us-gaapSegmentReportingInformationLineItems" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_ScheduleOfSegmentReportingInformationBySegmentTable" xlink:label="loc_us-gaapScheduleOfSegmentReportingInformationBySegmentTable" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xbrldt:contextElement="segment" xlink:from="loc_us-gaapSegmentReportingInformationLineItems" xlink:to="loc_us-gaapScheduleOfSegmentReportingInformationBySegmentTable" xlink:type="arc" order="10" xbrldt:closed="true" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/srt/2024/elts/srt-2024.xsd#srt_ConsolidationItemsAxis" xlink:label="loc_srtConsolidationItemsAxis" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_us-gaapScheduleOfSegmentReportingInformationBySegmentTable" xlink:to="loc_srtConsolidationItemsAxis" xlink:type="arc" order="1" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/srt/2024/elts/srt-2024.xsd#srt_ConsolidationItemsDomain" xlink:label="loc_srtConsolidationItemsDomain" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_srtConsolidationItemsAxis" xlink:to="loc_srtConsolidationItemsDomain" xlink:type="arc" order="20" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/srt/2024/elts/srt-2024.xsd#srt_ConsolidationItemsDomain" xlink:label="loc_srtConsolidationItemsDomain_10" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xbrldt:contextElement="segment" xlink:from="loc_srtConsolidationItemsAxis" xlink:to="loc_srtConsolidationItemsDomain_10" xlink:type="arc" order="30" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_OperatingSegmentsMember" xlink:label="loc_us-gaapOperatingSegmentsMember_0" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_srtConsolidationItemsDomain" xlink:to="loc_us-gaapOperatingSegmentsMember_0" xlink:type="arc" order="1" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.sec.gov/dei/2024/dei-2024.xsd#dei_LegalEntityAxis" xlink:label="loc_deiLegalEntityAxis" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_us-gaapScheduleOfSegmentReportingInformationBySegmentTable" xlink:to="loc_deiLegalEntityAxis" xlink:type="arc" order="2" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.sec.gov/dei/2024/dei-2024.xsd#dei_EntityDomain" xlink:label="loc_deiEntityDomain" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_deiLegalEntityAxis" xlink:to="loc_deiEntityDomain" xlink:type="arc" order="20" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.sec.gov/dei/2024/dei-2024.xsd#dei_EntityDomain" xlink:label="loc_deiEntityDomain_10" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xbrldt:contextElement="segment" xlink:from="loc_deiLegalEntityAxis" xlink:to="loc_deiEntityDomain_10" xlink:type="arc" order="30" />
      <link:loc xlink:type="locator" xlink:href="nito-20241231.xsd#NITO_PathogenPreventionAndProlongShelfLifeMember" xlink:label="loc_NITOPathogenPreventionAndProlongShelfLifeMember_0" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_deiEntityDomain" xlink:to="loc_NITOPathogenPreventionAndProlongShelfLifeMember_0" xlink:type="arc" order="2" />
      <link:loc xlink:type="locator" xlink:href="nito-20241231.xsd#NITO_N2OEmissionsGlobalWarmingSolutionsMember" xlink:label="loc_NITON2OEmissionsGlobalWarmingSolutionsMember_20" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_deiEntityDomain" xlink:to="loc_NITON2OEmissionsGlobalWarmingSolutionsMember_20" xlink:type="arc" order="22" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_RevenueFromContractWithCustomerExcludingAssessedTax" xlink:label="loc_us-gaapRevenueFromContractWithCustomerExcludingAssessedTax_70" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapSegmentReportingInformationLineItems" xlink:to="loc_us-gaapRevenueFromContractWithCustomerExcludingAssessedTax_70" xlink:type="arc" order="0" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_CostOfGoodsAndServicesSold" xlink:label="loc_us-gaapCostOfGoodsAndServicesSold_70" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapSegmentReportingInformationLineItems" xlink:to="loc_us-gaapCostOfGoodsAndServicesSold_70" xlink:type="arc" order="1" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_OperatingExpenses" xlink:label="loc_us-gaapOperatingExpenses_70" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapSegmentReportingInformationLineItems" xlink:to="loc_us-gaapOperatingExpenses_70" xlink:type="arc" order="2" />
      <link:loc xlink:type="locator" xlink:href="nito-20241231.xsd#NITO_ProfessionalServices" xlink:label="loc_NITOProfessionalServices_70" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapSegmentReportingInformationLineItems" xlink:to="loc_NITOProfessionalServices_70" xlink:type="arc" order="3" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_ShareBasedCompensation" xlink:label="loc_us-gaapShareBasedCompensation_70" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapSegmentReportingInformationLineItems" xlink:to="loc_us-gaapShareBasedCompensation_70" xlink:type="arc" order="4" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_Depreciation" xlink:label="loc_us-gaapDepreciation_70" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapSegmentReportingInformationLineItems" xlink:to="loc_us-gaapDepreciation_70" xlink:type="arc" order="5" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_OtherGeneralAndAdministrativeExpense" xlink:label="loc_us-gaapOtherGeneralAndAdministrativeExpense_70" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapSegmentReportingInformationLineItems" xlink:to="loc_us-gaapOtherGeneralAndAdministrativeExpense_70" xlink:type="arc" order="6" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_OperatingIncomeLoss" xlink:label="loc_us-gaapOperatingIncomeLoss_70" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapSegmentReportingInformationLineItems" xlink:to="loc_us-gaapOperatingIncomeLoss_70" xlink:type="arc" order="7" />
      <link:loc xlink:type="locator" xlink:href="nito-20241231.xsd#NITO_FinancingExpensesIncomeNet" xlink:label="loc_NITOFinancingExpensesIncomeNet_70" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapSegmentReportingInformationLineItems" xlink:to="loc_NITOFinancingExpensesIncomeNet_70" xlink:type="arc" order="8" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_OtherNonoperatingIncome" xlink:label="loc_us-gaapOtherNonoperatingIncome_70" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapSegmentReportingInformationLineItems" xlink:to="loc_us-gaapOtherNonoperatingIncome_70" xlink:type="arc" order="9" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_DebtSecuritiesUnrealizedGainLoss" xlink:label="loc_us-gaapDebtSecuritiesUnrealizedGainLoss_70" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapSegmentReportingInformationLineItems" xlink:to="loc_us-gaapDebtSecuritiesUnrealizedGainLoss_70" xlink:type="arc" order="10" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_ProfitLoss" xlink:label="loc_us-gaapProfitLoss_70" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapSegmentReportingInformationLineItems" xlink:to="loc_us-gaapProfitLoss_70" xlink:type="arc" order="11" />
    </link:definitionLink>
    <link:definitionLink xlink:type="extended" xlink:role="http://n2off.com/role/ScheduleOfInformationOnSalesByGeographicDistributionDetails" xlink:title="999097 - Disclosure - SCHEDULE OF INFORMATION ON SALES BY GEOGRAPHIC DISTRIBUTION (Details)">
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_RevenuesFromExternalCustomersAndLongLivedAssetsLineItems" xlink:label="loc_us-gaapRevenuesFromExternalCustomersAndLongLivedAssetsLineItems" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_ScheduleOfRevenuesFromExternalCustomersAndLongLivedAssetsTable" xlink:label="loc_us-gaapScheduleOfRevenuesFromExternalCustomersAndLongLivedAssetsTable" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xbrldt:contextElement="segment" xlink:from="loc_us-gaapRevenuesFromExternalCustomersAndLongLivedAssetsLineItems" xlink:to="loc_us-gaapScheduleOfRevenuesFromExternalCustomersAndLongLivedAssetsTable" xlink:type="arc" order="10" xbrldt:closed="true" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/srt/2024/elts/srt-2024.xsd#srt_StatementGeographicalAxis" xlink:label="loc_srtStatementGeographicalAxis" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_us-gaapScheduleOfRevenuesFromExternalCustomersAndLongLivedAssetsTable" xlink:to="loc_srtStatementGeographicalAxis" xlink:type="arc" order="1" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/srt/2024/elts/srt-2024.xsd#srt_SegmentGeographicalDomain" xlink:label="loc_srtSegmentGeographicalDomain" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_srtStatementGeographicalAxis" xlink:to="loc_srtSegmentGeographicalDomain" xlink:type="arc" order="20" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/srt/2024/elts/srt-2024.xsd#srt_SegmentGeographicalDomain" xlink:label="loc_srtSegmentGeographicalDomain_10" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xbrldt:contextElement="segment" xlink:from="loc_srtStatementGeographicalAxis" xlink:to="loc_srtSegmentGeographicalDomain_10" xlink:type="arc" order="30" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.sec.gov/country/2024/country-2024.xsd#country_US" xlink:label="loc_countryUS_0" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_srtSegmentGeographicalDomain" xlink:to="loc_countryUS_0" xlink:type="arc" order="1" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.sec.gov/country/2024/country-2024.xsd#country_MX" xlink:label="loc_countryMX_20" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_srtSegmentGeographicalDomain" xlink:to="loc_countryMX_20" xlink:type="arc" order="21" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.sec.gov/country/2024/country-2024.xsd#country_IL" xlink:label="loc_countryIL_40" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_srtSegmentGeographicalDomain" xlink:to="loc_countryIL_40" xlink:type="arc" order="41" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.sec.gov/country/2024/country-2024.xsd#country_PE" xlink:label="loc_countryPE_60" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_srtSegmentGeographicalDomain" xlink:to="loc_countryPE_60" xlink:type="arc" order="61" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.sec.gov/country/2024/country-2024.xsd#country_TR" xlink:label="loc_countryTR_80" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_srtSegmentGeographicalDomain" xlink:to="loc_countryTR_80" xlink:type="arc" order="81" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_RevenueFromContractWithCustomerExcludingAssessedTax" xlink:label="loc_us-gaapRevenueFromContractWithCustomerExcludingAssessedTax_130" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapRevenuesFromExternalCustomersAndLongLivedAssetsLineItems" xlink:to="loc_us-gaapRevenueFromContractWithCustomerExcludingAssessedTax_130" xlink:type="arc" order="0" />
    </link:definitionLink>
    <link:definitionLink xlink:type="extended" xlink:role="http://n2off.com/role/ScheduleOfSalesToCustomersDetails" xlink:title="999098 - Disclosure - SCHEDULE OF SALES TO CUSTOMERS (Details)">
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_EntityWideRevenueMajorCustomerLineItems" xlink:label="loc_us-gaapEntityWideRevenueMajorCustomerLineItems" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_ScheduleOfEntityWideRevenueByMajorCustomersByReportingSegmentsTable" xlink:label="loc_us-gaapScheduleOfEntityWideRevenueByMajorCustomersByReportingSegmentsTable" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xbrldt:contextElement="segment" xlink:from="loc_us-gaapEntityWideRevenueMajorCustomerLineItems" xlink:to="loc_us-gaapScheduleOfEntityWideRevenueByMajorCustomersByReportingSegmentsTable" xlink:type="arc" order="10" xbrldt:closed="true" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/srt/2024/elts/srt-2024.xsd#srt_MajorCustomersAxis" xlink:label="loc_srtMajorCustomersAxis" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_us-gaapScheduleOfEntityWideRevenueByMajorCustomersByReportingSegmentsTable" xlink:to="loc_srtMajorCustomersAxis" xlink:type="arc" order="1" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/srt/2024/elts/srt-2024.xsd#srt_NameOfMajorCustomerDomain" xlink:label="loc_srtNameOfMajorCustomerDomain" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_srtMajorCustomersAxis" xlink:to="loc_srtNameOfMajorCustomerDomain" xlink:type="arc" order="20" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/srt/2024/elts/srt-2024.xsd#srt_NameOfMajorCustomerDomain" xlink:label="loc_srtNameOfMajorCustomerDomain_10" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xbrldt:contextElement="segment" xlink:from="loc_srtMajorCustomersAxis" xlink:to="loc_srtNameOfMajorCustomerDomain_10" xlink:type="arc" order="30" />
      <link:loc xlink:type="locator" xlink:href="nito-20241231.xsd#NITO_CustomerAMember" xlink:label="loc_NITOCustomerAMember_0" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_srtNameOfMajorCustomerDomain" xlink:to="loc_NITOCustomerAMember_0" xlink:type="arc" order="1" />
      <link:loc xlink:type="locator" xlink:href="nito-20241231.xsd#NITO_CustomerBMember" xlink:label="loc_NITOCustomerBMember_20" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_srtNameOfMajorCustomerDomain" xlink:to="loc_NITOCustomerBMember_20" xlink:type="arc" order="21" />
      <link:loc xlink:type="locator" xlink:href="nito-20241231.xsd#NITO_CustomerCMember" xlink:label="loc_NITOCustomerCMember_40" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_srtNameOfMajorCustomerDomain" xlink:to="loc_NITOCustomerCMember_40" xlink:type="arc" order="41" />
      <link:loc xlink:type="locator" xlink:href="nito-20241231.xsd#NITO_CustomersMember" xlink:label="loc_NITOCustomersMember_60" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_srtNameOfMajorCustomerDomain" xlink:to="loc_NITOCustomersMember_60" xlink:type="arc" order="61" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_RevenueFromContractWithCustomerExcludingAssessedTax" xlink:label="loc_us-gaapRevenueFromContractWithCustomerExcludingAssessedTax_90" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapEntityWideRevenueMajorCustomerLineItems" xlink:to="loc_us-gaapRevenueFromContractWithCustomerExcludingAssessedTax_90" xlink:type="arc" order="0" />
    </link:definitionLink>
    <link:definitionLink xlink:type="extended" xlink:role="http://n2off.com/role/ScheduleOfInformationOnLongLivedAssetsDetails" xlink:title="999099 - Disclosure - SCHEDULE OF INFORMATION ON LONG LIVED ASSETS (Details)">
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_RevenuesFromExternalCustomersAndLongLivedAssetsLineItems" xlink:label="loc_us-gaapRevenuesFromExternalCustomersAndLongLivedAssetsLineItems" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_ScheduleOfRevenuesFromExternalCustomersAndLongLivedAssetsTable" xlink:label="loc_us-gaapScheduleOfRevenuesFromExternalCustomersAndLongLivedAssetsTable" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xbrldt:contextElement="segment" xlink:from="loc_us-gaapRevenuesFromExternalCustomersAndLongLivedAssetsLineItems" xlink:to="loc_us-gaapScheduleOfRevenuesFromExternalCustomersAndLongLivedAssetsTable" xlink:type="arc" order="10" xbrldt:closed="true" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/srt/2024/elts/srt-2024.xsd#srt_StatementGeographicalAxis" xlink:label="loc_srtStatementGeographicalAxis" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_us-gaapScheduleOfRevenuesFromExternalCustomersAndLongLivedAssetsTable" xlink:to="loc_srtStatementGeographicalAxis" xlink:type="arc" order="1" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/srt/2024/elts/srt-2024.xsd#srt_SegmentGeographicalDomain" xlink:label="loc_srtSegmentGeographicalDomain" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_srtStatementGeographicalAxis" xlink:to="loc_srtSegmentGeographicalDomain" xlink:type="arc" order="20" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/srt/2024/elts/srt-2024.xsd#srt_SegmentGeographicalDomain" xlink:label="loc_srtSegmentGeographicalDomain_10" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xbrldt:contextElement="segment" xlink:from="loc_srtStatementGeographicalAxis" xlink:to="loc_srtSegmentGeographicalDomain_10" xlink:type="arc" order="30" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.sec.gov/country/2024/country-2024.xsd#country_IL" xlink:label="loc_countryIL_0" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_srtSegmentGeographicalDomain" xlink:to="loc_countryIL_0" xlink:type="arc" order="1" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.sec.gov/country/2024/country-2024.xsd#country_US" xlink:label="loc_countryUS_20" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_srtSegmentGeographicalDomain" xlink:to="loc_countryUS_20" xlink:type="arc" order="21" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_NoncurrentAssets" xlink:label="loc_us-gaapNoncurrentAssets_70" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapRevenuesFromExternalCustomersAndLongLivedAssetsLineItems" xlink:to="loc_us-gaapNoncurrentAssets_70" xlink:type="arc" order="0" />
    </link:definitionLink>
    <link:definitionLink xlink:type="extended" xlink:role="http://n2off.com/role/SegmentReportingDetailsNarrative" xlink:title="999100 - Disclosure - SEGMENT REPORTING (Details Narrative)">
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_EntityWideRevenueMajorCustomerLineItems" xlink:label="loc_us-gaapEntityWideRevenueMajorCustomerLineItems" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_ScheduleOfEntityWideRevenueByMajorCustomersByReportingSegmentsTable" xlink:label="loc_us-gaapScheduleOfEntityWideRevenueByMajorCustomersByReportingSegmentsTable" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xbrldt:contextElement="segment" xlink:from="loc_us-gaapEntityWideRevenueMajorCustomerLineItems" xlink:to="loc_us-gaapScheduleOfEntityWideRevenueByMajorCustomersByReportingSegmentsTable" xlink:type="arc" order="10" xbrldt:closed="true" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/srt/2024/elts/srt-2024.xsd#srt_MajorCustomersAxis" xlink:label="loc_srtMajorCustomersAxis" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_us-gaapScheduleOfEntityWideRevenueByMajorCustomersByReportingSegmentsTable" xlink:to="loc_srtMajorCustomersAxis" xlink:type="arc" order="1" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/srt/2024/elts/srt-2024.xsd#srt_NameOfMajorCustomerDomain" xlink:label="loc_srtNameOfMajorCustomerDomain" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_srtMajorCustomersAxis" xlink:to="loc_srtNameOfMajorCustomerDomain" xlink:type="arc" order="20" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/srt/2024/elts/srt-2024.xsd#srt_NameOfMajorCustomerDomain" xlink:label="loc_srtNameOfMajorCustomerDomain_10" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xbrldt:contextElement="segment" xlink:from="loc_srtMajorCustomersAxis" xlink:to="loc_srtNameOfMajorCustomerDomain_10" xlink:type="arc" order="30" />
      <link:loc xlink:type="locator" xlink:href="nito-20241231.xsd#NITO_SingleCustomerMember" xlink:label="loc_NITOSingleCustomerMember_0" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_srtNameOfMajorCustomerDomain" xlink:to="loc_NITOSingleCustomerMember_0" xlink:type="arc" order="1" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_ConcentrationRiskByBenchmarkAxis" xlink:label="loc_us-gaapConcentrationRiskByBenchmarkAxis" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_us-gaapScheduleOfEntityWideRevenueByMajorCustomersByReportingSegmentsTable" xlink:to="loc_us-gaapConcentrationRiskByBenchmarkAxis" xlink:type="arc" order="2" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_ConcentrationRiskBenchmarkDomain" xlink:label="loc_us-gaapConcentrationRiskBenchmarkDomain" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_us-gaapConcentrationRiskByBenchmarkAxis" xlink:to="loc_us-gaapConcentrationRiskBenchmarkDomain" xlink:type="arc" order="20" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_ConcentrationRiskBenchmarkDomain" xlink:label="loc_us-gaapConcentrationRiskBenchmarkDomain_10" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xbrldt:contextElement="segment" xlink:from="loc_us-gaapConcentrationRiskByBenchmarkAxis" xlink:to="loc_us-gaapConcentrationRiskBenchmarkDomain_10" xlink:type="arc" order="30" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_SalesRevenueSegmentMember" xlink:label="loc_us-gaapSalesRevenueSegmentMember_0" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapConcentrationRiskBenchmarkDomain" xlink:to="loc_us-gaapSalesRevenueSegmentMember_0" xlink:type="arc" order="2" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_ConcentrationRiskByTypeAxis" xlink:label="loc_us-gaapConcentrationRiskByTypeAxis" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_us-gaapScheduleOfEntityWideRevenueByMajorCustomersByReportingSegmentsTable" xlink:to="loc_us-gaapConcentrationRiskByTypeAxis" xlink:type="arc" order="3" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_ConcentrationRiskTypeDomain" xlink:label="loc_us-gaapConcentrationRiskTypeDomain" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_us-gaapConcentrationRiskByTypeAxis" xlink:to="loc_us-gaapConcentrationRiskTypeDomain" xlink:type="arc" order="20" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_ConcentrationRiskTypeDomain" xlink:label="loc_us-gaapConcentrationRiskTypeDomain_10" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xbrldt:contextElement="segment" xlink:from="loc_us-gaapConcentrationRiskByTypeAxis" xlink:to="loc_us-gaapConcentrationRiskTypeDomain_10" xlink:type="arc" order="30" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_CustomerConcentrationRiskMember" xlink:label="loc_us-gaapCustomerConcentrationRiskMember_0" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapConcentrationRiskTypeDomain" xlink:to="loc_us-gaapCustomerConcentrationRiskMember_0" xlink:type="arc" order="3" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_ConcentrationRiskPercentage1" xlink:label="loc_us-gaapConcentrationRiskPercentage1_20" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapEntityWideRevenueMajorCustomerLineItems" xlink:to="loc_us-gaapConcentrationRiskPercentage1_20" xlink:type="arc" order="0" />
    </link:definitionLink>
    <link:definitionLink xlink:type="extended" xlink:role="http://n2off.com/role/SubsequentEventsDetailsNarrative" xlink:title="999101 - Disclosure - SUBSEQUENT EVENTS (Details Narrative)">
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_SubsequentEventLineItems" xlink:label="loc_us-gaapSubsequentEventLineItems" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_SubsequentEventTable" xlink:label="loc_us-gaapSubsequentEventTable" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xbrldt:contextElement="segment" xlink:from="loc_us-gaapSubsequentEventLineItems" xlink:to="loc_us-gaapSubsequentEventTable" xlink:type="arc" order="10" xbrldt:closed="true" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_SubsequentEventTypeAxis" xlink:label="loc_us-gaapSubsequentEventTypeAxis" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_us-gaapSubsequentEventTable" xlink:to="loc_us-gaapSubsequentEventTypeAxis" xlink:type="arc" order="1" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_SubsequentEventTypeDomain" xlink:label="loc_us-gaapSubsequentEventTypeDomain" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_us-gaapSubsequentEventTypeAxis" xlink:to="loc_us-gaapSubsequentEventTypeDomain" xlink:type="arc" order="20" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_SubsequentEventTypeDomain" xlink:label="loc_us-gaapSubsequentEventTypeDomain_10" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xbrldt:contextElement="segment" xlink:from="loc_us-gaapSubsequentEventTypeAxis" xlink:to="loc_us-gaapSubsequentEventTypeDomain_10" xlink:type="arc" order="30" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_SubsequentEventMember" xlink:label="loc_us-gaapSubsequentEventMember_0" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapSubsequentEventTypeDomain" xlink:to="loc_us-gaapSubsequentEventMember_0" xlink:type="arc" order="1" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_SubsidiarySaleOfStockAxis" xlink:label="loc_us-gaapSubsidiarySaleOfStockAxis" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_us-gaapSubsequentEventTable" xlink:to="loc_us-gaapSubsidiarySaleOfStockAxis" xlink:type="arc" order="2" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_SaleOfStockNameOfTransactionDomain" xlink:label="loc_us-gaapSaleOfStockNameOfTransactionDomain" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_us-gaapSubsidiarySaleOfStockAxis" xlink:to="loc_us-gaapSaleOfStockNameOfTransactionDomain" xlink:type="arc" order="20" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_SaleOfStockNameOfTransactionDomain" xlink:label="loc_us-gaapSaleOfStockNameOfTransactionDomain_10" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xbrldt:contextElement="segment" xlink:from="loc_us-gaapSubsidiarySaleOfStockAxis" xlink:to="loc_us-gaapSaleOfStockNameOfTransactionDomain_10" xlink:type="arc" order="30" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_PrivatePlacementMember" xlink:label="loc_us-gaapPrivatePlacementMember_0" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapSaleOfStockNameOfTransactionDomain" xlink:to="loc_us-gaapPrivatePlacementMember_0" xlink:type="arc" order="2" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_TypeOfArrangementAxis" xlink:label="loc_us-gaapTypeOfArrangementAxis" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_us-gaapSubsequentEventTable" xlink:to="loc_us-gaapTypeOfArrangementAxis" xlink:type="arc" order="3" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_ArrangementsAndNonarrangementTransactionsMember" xlink:label="loc_us-gaapArrangementsAndNonarrangementTransactionsMember" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_us-gaapTypeOfArrangementAxis" xlink:to="loc_us-gaapArrangementsAndNonarrangementTransactionsMember" xlink:type="arc" order="20" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_ArrangementsAndNonarrangementTransactionsMember" xlink:label="loc_us-gaapArrangementsAndNonarrangementTransactionsMember_10" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xbrldt:contextElement="segment" xlink:from="loc_us-gaapTypeOfArrangementAxis" xlink:to="loc_us-gaapArrangementsAndNonarrangementTransactionsMember_10" xlink:type="arc" order="30" />
      <link:loc xlink:type="locator" xlink:href="nito-20241231.xsd#NITO_SecuritiesPurchaseAgreementMember" xlink:label="loc_NITOSecuritiesPurchaseAgreementMember_0" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapArrangementsAndNonarrangementTransactionsMember" xlink:to="loc_NITOSecuritiesPurchaseAgreementMember_0" xlink:type="arc" order="3" />
      <link:loc xlink:type="locator" xlink:href="nito-20241231.xsd#NITO_ItalyAgreementMember" xlink:label="loc_NITOItalyAgreementMember_90" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapArrangementsAndNonarrangementTransactionsMember" xlink:to="loc_NITOItalyAgreementMember_90" xlink:type="arc" order="92" />
      <link:loc xlink:type="locator" xlink:href="nito-20241231.xsd#NITO_MitoAgreementMember" xlink:label="loc_NITOMitoAgreementMember_120" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapArrangementsAndNonarrangementTransactionsMember" xlink:to="loc_NITOMitoAgreementMember_120" xlink:type="arc" order="122" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_StatementEquityComponentsAxis" xlink:label="loc_us-gaapStatementEquityComponentsAxis" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_us-gaapSubsequentEventTable" xlink:to="loc_us-gaapStatementEquityComponentsAxis" xlink:type="arc" order="4" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_EquityComponentDomain" xlink:label="loc_us-gaapEquityComponentDomain" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_us-gaapStatementEquityComponentsAxis" xlink:to="loc_us-gaapEquityComponentDomain" xlink:type="arc" order="20" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_EquityComponentDomain" xlink:label="loc_us-gaapEquityComponentDomain_10" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xbrldt:contextElement="segment" xlink:from="loc_us-gaapStatementEquityComponentsAxis" xlink:to="loc_us-gaapEquityComponentDomain_10" xlink:type="arc" order="30" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_CommonStockMember" xlink:label="loc_us-gaapCommonStockMember_20" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapEquityComponentDomain" xlink:to="loc_us-gaapCommonStockMember_20" xlink:type="arc" order="22" />
      <link:loc xlink:type="locator" xlink:href="nito-20241231.xsd#NITO_PreFundedWarrantsMember" xlink:label="loc_NITOPreFundedWarrantsMember_30" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapEquityComponentDomain" xlink:to="loc_NITOPreFundedWarrantsMember_30" xlink:type="arc" order="32" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/srt/2024/elts/srt-2024.xsd#srt_TitleOfIndividualAxis" xlink:label="loc_srtTitleOfIndividualAxis" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_us-gaapSubsequentEventTable" xlink:to="loc_srtTitleOfIndividualAxis" xlink:type="arc" order="5" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/srt/2024/elts/srt-2024.xsd#srt_TitleOfIndividualWithRelationshipToEntityDomain" xlink:label="loc_srtTitleOfIndividualWithRelationshipToEntityDomain" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_srtTitleOfIndividualAxis" xlink:to="loc_srtTitleOfIndividualWithRelationshipToEntityDomain" xlink:type="arc" order="20" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/srt/2024/elts/srt-2024.xsd#srt_TitleOfIndividualWithRelationshipToEntityDomain" xlink:label="loc_srtTitleOfIndividualWithRelationshipToEntityDomain_10" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xbrldt:contextElement="segment" xlink:from="loc_srtTitleOfIndividualAxis" xlink:to="loc_srtTitleOfIndividualWithRelationshipToEntityDomain_10" xlink:type="arc" order="30" />
      <link:loc xlink:type="locator" xlink:href="nito-20241231.xsd#NITO_AlonMember" xlink:label="loc_NITOAlonMember_140" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_srtTitleOfIndividualWithRelationshipToEntityDomain" xlink:to="loc_NITOAlonMember_140" xlink:type="arc" order="143" />
      <link:loc xlink:type="locator" xlink:href="nito-20241231.xsd#NITO_CiroMember" xlink:label="loc_NITOCiroMember_150" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_srtTitleOfIndividualWithRelationshipToEntityDomain" xlink:to="loc_NITOCiroMember_150" xlink:type="arc" order="153" />
      <link:loc xlink:type="locator" xlink:href="nito-20241231.xsd#NITO_SciSparcMember" xlink:label="loc_NITOSciSparcMember_160" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_srtTitleOfIndividualWithRelationshipToEntityDomain" xlink:to="loc_NITOSciSparcMember_160" xlink:type="arc" order="163" />
      <link:loc xlink:type="locator" xlink:href="nito-20241231.xsd#NITO_MitoCareXMember" xlink:label="loc_NITOMitoCareXMember_220" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_srtTitleOfIndividualWithRelationshipToEntityDomain" xlink:to="loc_NITOMitoCareXMember_220" xlink:type="arc" order="223" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/srt/2024/elts/srt-2024.xsd#srt_OwnershipAxis" xlink:label="loc_srtOwnershipAxis" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_us-gaapSubsequentEventTable" xlink:to="loc_srtOwnershipAxis" xlink:type="arc" order="6" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/srt/2024/elts/srt-2024.xsd#srt_OwnershipDomain" xlink:label="loc_srtOwnershipDomain" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_srtOwnershipAxis" xlink:to="loc_srtOwnershipDomain" xlink:type="arc" order="20" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/srt/2024/elts/srt-2024.xsd#srt_OwnershipDomain" xlink:label="loc_srtOwnershipDomain_10" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xbrldt:contextElement="segment" xlink:from="loc_srtOwnershipAxis" xlink:to="loc_srtOwnershipDomain_10" xlink:type="arc" order="30" />
      <link:loc xlink:type="locator" xlink:href="nito-20241231.xsd#NITO_SciSparcMember" xlink:label="loc_NITOSciSparcMember_170" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_srtOwnershipDomain" xlink:to="loc_NITOSciSparcMember_170" xlink:type="arc" order="173" />
      <link:loc xlink:type="locator" xlink:href="nito-20241231.xsd#NITO_MitoAgreementMember" xlink:label="loc_NITOMitoAgreementMember_180" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_srtOwnershipDomain" xlink:to="loc_NITOMitoAgreementMember_180" xlink:type="arc" order="182" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/srt/2024/elts/srt-2024.xsd#srt_RangeAxis" xlink:label="loc_srtRangeAxis" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_us-gaapSubsequentEventTable" xlink:to="loc_srtRangeAxis" xlink:type="arc" order="7" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/srt/2024/elts/srt-2024.xsd#srt_RangeMember" xlink:label="loc_srtRangeMember" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_srtRangeAxis" xlink:to="loc_srtRangeMember" xlink:type="arc" order="20" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/srt/2024/elts/srt-2024.xsd#srt_RangeMember" xlink:label="loc_srtRangeMember_10" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xbrldt:contextElement="segment" xlink:from="loc_srtRangeAxis" xlink:to="loc_srtRangeMember_10" xlink:type="arc" order="30" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/srt/2024/elts/srt-2024.xsd#srt_MaximumMember" xlink:label="loc_srtMaximumMember_190" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_srtRangeMember" xlink:to="loc_srtMaximumMember_190" xlink:type="arc" order="192" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_StockIssuedDuringPeriodSharesNewIssues" xlink:label="loc_us-gaapStockIssuedDuringPeriodSharesNewIssues_250" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapSubsequentEventLineItems" xlink:to="loc_us-gaapStockIssuedDuringPeriodSharesNewIssues_250" xlink:type="arc" order="0" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_ClassOfWarrantOrRightNumberOfSecuritiesCalledByWarrantsOrRights" xlink:label="loc_us-gaapClassOfWarrantOrRightNumberOfSecuritiesCalledByWarrantsOrRights_250" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapSubsequentEventLineItems" xlink:to="loc_us-gaapClassOfWarrantOrRightNumberOfSecuritiesCalledByWarrantsOrRights_250" xlink:type="arc" order="1" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_ClassOfWarrantOrRightExercisePriceOfWarrantsOrRights1" xlink:label="loc_us-gaapClassOfWarrantOrRightExercisePriceOfWarrantsOrRights1_250" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapSubsequentEventLineItems" xlink:to="loc_us-gaapClassOfWarrantOrRightExercisePriceOfWarrantsOrRights1_250" xlink:type="arc" order="2" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_WarrantsAndRightsOutstanding" xlink:label="loc_us-gaapWarrantsAndRightsOutstanding_250" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapSubsequentEventLineItems" xlink:to="loc_us-gaapWarrantsAndRightsOutstanding_250" xlink:type="arc" order="3" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardNonOptionEquityInstrumentsExercised" xlink:label="loc_us-gaapShareBasedCompensationArrangementByShareBasedPaymentAwardNonOptionEquityInstrumentsExercised_250" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapSubsequentEventLineItems" xlink:to="loc_us-gaapShareBasedCompensationArrangementByShareBasedPaymentAwardNonOptionEquityInstrumentsExercised_250" xlink:type="arc" order="4" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_ProceedsFromIssuanceOfWarrants" xlink:label="loc_us-gaapProceedsFromIssuanceOfWarrants_250" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapSubsequentEventLineItems" xlink:to="loc_us-gaapProceedsFromIssuanceOfWarrants_250" xlink:type="arc" order="5" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_ProceedsFromLinesOfCredit" xlink:label="loc_us-gaapProceedsFromLinesOfCredit_250" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapSubsequentEventLineItems" xlink:to="loc_us-gaapProceedsFromLinesOfCredit_250" xlink:type="arc" order="6" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_SaleOfStockPercentageOfOwnershipAfterTransaction" xlink:label="loc_us-gaapSaleOfStockPercentageOfOwnershipAfterTransaction_250" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapSubsequentEventLineItems" xlink:to="loc_us-gaapSaleOfStockPercentageOfOwnershipAfterTransaction_250" xlink:type="arc" order="7" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_DebtCurrent" xlink:label="loc_us-gaapDebtCurrent_250" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapSubsequentEventLineItems" xlink:to="loc_us-gaapDebtCurrent_250" xlink:type="arc" order="8" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_DebtInstrumentInterestRateEffectivePercentage" xlink:label="loc_us-gaapDebtInstrumentInterestRateEffectivePercentage_250" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapSubsequentEventLineItems" xlink:to="loc_us-gaapDebtInstrumentInterestRateEffectivePercentage_250" xlink:type="arc" order="9" />
      <link:loc xlink:type="locator" xlink:href="nito-20241231.xsd#NITO_DescriptionForNetProfitFormSales" xlink:label="loc_NITODescriptionForNetProfitFormSales_250" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapSubsequentEventLineItems" xlink:to="loc_NITODescriptionForNetProfitFormSales_250" xlink:type="arc" order="10" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_CommonStockParOrStatedValuePerShare" xlink:label="loc_us-gaapCommonStockParOrStatedValuePerShare_250" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapSubsequentEventLineItems" xlink:to="loc_us-gaapCommonStockParOrStatedValuePerShare_250" xlink:type="arc" order="11" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_SaleOfStockNumberOfSharesIssuedInTransaction" xlink:label="loc_us-gaapSaleOfStockNumberOfSharesIssuedInTransaction_250" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapSubsequentEventLineItems" xlink:to="loc_us-gaapSaleOfStockNumberOfSharesIssuedInTransaction_250" xlink:type="arc" order="12" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_PaymentsOfStockIssuanceCosts" xlink:label="loc_us-gaapPaymentsOfStockIssuanceCosts_250" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapSubsequentEventLineItems" xlink:to="loc_us-gaapPaymentsOfStockIssuanceCosts_250" xlink:type="arc" order="13" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_SaleOfStockPercentageOfOwnershipBeforeTransaction" xlink:label="loc_us-gaapSaleOfStockPercentageOfOwnershipBeforeTransaction_250" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapSubsequentEventLineItems" xlink:to="loc_us-gaapSaleOfStockPercentageOfOwnershipBeforeTransaction_250" xlink:type="arc" order="14" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_MinorityInterestOwnershipPercentageByParent" xlink:label="loc_us-gaapMinorityInterestOwnershipPercentageByParent_250" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapSubsequentEventLineItems" xlink:to="loc_us-gaapMinorityInterestOwnershipPercentageByParent_250" xlink:type="arc" order="15" />
      <link:loc xlink:type="locator" xlink:href="nito-20241231.xsd#NITO_GrantOfRestrictedStockPercentage" xlink:label="loc_NITOGrantOfRestrictedStockPercentage_250" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapSubsequentEventLineItems" xlink:to="loc_NITOGrantOfRestrictedStockPercentage_250" xlink:type="arc" order="16" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_OtherNotesPayable" xlink:label="loc_us-gaapOtherNotesPayable_250" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapSubsequentEventLineItems" xlink:to="loc_us-gaapOtherNotesPayable_250" xlink:type="arc" order="17" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_Investments" xlink:label="loc_us-gaapInvestments_250" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapSubsequentEventLineItems" xlink:to="loc_us-gaapInvestments_250" xlink:type="arc" order="18" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_DebtInstrumentFaceAmount" xlink:label="loc_us-gaapDebtInstrumentFaceAmount_250" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapSubsequentEventLineItems" xlink:to="loc_us-gaapDebtInstrumentFaceAmount_250" xlink:type="arc" order="19" />
    </link:definitionLink>
    <link:definitionLink xlink:type="extended" xlink:role="http://xbrl.sec.gov/ecd/role/PvpExecutiveCategoriesOnly" />
    <link:definitionLink xlink:type="extended" xlink:role="http://xbrl.sec.gov/ecd/role/PvpAdjustmentsOnly" />
    <link:definitionLink xlink:type="extended" xlink:role="http://xbrl.sec.gov/ecd/role/IndividualsOnly" />
    <link:definitionLink xlink:type="extended" xlink:role="http://xbrl.sec.gov/ecd/AwardTypeOnly">
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2024/elts/us-gaap-2024.xsd#us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain" xlink:label="loc_us-gaapShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain" />
      <link:loc xlink:type="locator" xlink:href="nito-20241231.xsd#NITO_FirstOptionPeriodMember" xlink:label="loc_NITOFirstOptionPeriodMember" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain" xlink:to="loc_NITOFirstOptionPeriodMember" xlink:type="arc" order="0" />
      <link:loc xlink:type="locator" xlink:href="nito-20241231.xsd#NITO_SecondOptionPeriodMember" xlink:label="loc_NITOSecondOptionPeriodMember" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain" xlink:to="loc_NITOSecondOptionPeriodMember" xlink:type="arc" order="10" />
      <link:loc xlink:type="locator" xlink:href="nito-20241231.xsd#NITO_AdditionalOptionPeriodMember" xlink:label="loc_NITOAdditionalOptionPeriodMember" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain" xlink:to="loc_NITOAdditionalOptionPeriodMember" xlink:type="arc" order="20" />
      <link:loc xlink:type="locator" xlink:href="nito-20241231.xsd#NITO_SixMonthsAnniversaryMember" xlink:label="loc_NITOSixMonthsAnniversaryMember" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain" xlink:to="loc_NITOSixMonthsAnniversaryMember" xlink:type="arc" order="30" />
      <link:loc xlink:type="locator" xlink:href="nito-20241231.xsd#NITO_NineMonthsAnniversaryMember" xlink:label="loc_NITONineMonthsAnniversaryMember" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain" xlink:to="loc_NITONineMonthsAnniversaryMember" xlink:type="arc" order="40" />
      <link:loc xlink:type="locator" xlink:href="nito-20241231.xsd#NITO_TwelveMonthsAnniversaryMember" xlink:label="loc_NITOTwelveMonthsAnniversaryMember" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain" xlink:to="loc_NITOTwelveMonthsAnniversaryMember" xlink:type="arc" order="50" />
      <link:loc xlink:type="locator" xlink:href="nito-20241231.xsd#NITO_ThreeMonthsMember" xlink:label="loc_NITOThreeMonthsMember" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain" xlink:to="loc_NITOThreeMonthsMember" xlink:type="arc" order="60" />
    </link:definitionLink>
    <link:definitionLink xlink:type="extended" xlink:role="http://xbrl.sec.gov/ecd/role/TradingArrangementsOnly" />
</link:linkbase>
