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Chemical investigations of L. stoechas subsp. luisieri and L. pedunculata essential oils were analyzed by GC-MS, and the antimicrobial activity was performed against bacteria and fungi isolated from food sources. The cytotoxicity of the essential oil was performed in NHDF cells using the MTT method. According to the results, the main compounds of L. stoechas subsp. luisieri essential oil were trans-α-necrodyl acetate (40.2%), lavandulyl acetate (11%), and trans-α-necrodol (10.4%), while fenchone (50.5%) and camphor (30.0%) in L. pedunculata essential oil. The antifungal activity of essential oils was confirmed with MIC values ranging from 1.2 to 18.7 µL/mL; for bacteria, it ranged from 4.7 to 149.3 µL/mL. Both the Lavandula species tested showed low or equal MIC and MBC/MFC values for L. stoechas subsp. luisieri essential oil, revealing greater efficacy in antimicrobial activity. The L. stoechas subsp. luisieri essential oil revealed cytotoxic effects (30 ± 2% of cell viability) in NHDF cells at all concentrations tested.