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Background & Aim: Due to the side effects of chemical and synthetic preservatives, consumers have recently become more eager to use foods containing natural preservatives from plants, animals and microbial sources. In the present study, biochemical composition and antibacterial effects (MIC) of Mentha longifolia L. essential oil against Staphylococcus aureus have been evaluated. Methods: In this experimental study, the biochemical composition and antibacterial prosperities of this essential oil was determined by the Gas chromatography/ mass spectrophotometer (GC/MS) and micro dilution method respectively. The morphological and membrane changes of the bacterial cell under the effect of this essential oil were evaluated by transmission electron microscopy. The collected data was analyzed by the SPSS software using ANOVA. Results: The chemical analysis of the essential oil by Gas chromatography/ mass spectrophotometer (GC/MS) revealed the presence of 22 substances (95.30%), mainly including Pulegon (31.54%), 1,8 Cineol (15.89%), Menthoforan (11.8%) and Cis- Isopulegon (9.74%). Minimum inhibitory concentration of the essential oil determined under different temperature and pH values showed to be in the range of 75-1200 µg/ ml. Conclusion: The MIC results and membrane cell damage observed in the electron microscopy evaluation indicated that this essential oil have a high antibacterial activity. Therefore, this essential oil can be combined with other agents for the preservation of foods against pathogenic and toxigenic microorganisms.