The aimof this studywas to determine the composition and antimicrobial effect ofMentha longifolia L. essential oil in Âin-vitroÂ condition. For this purpose, the chemical composition of the essential oil which obtained by hydrodistillation was examined by GC/MS and the antimicrobial effect was studied on the growth of bacterial species including Bacillus cereus, Pseudomonas aeruginosa and Proteus vulgaris using micro-dilution method. The minimuminhibitory concentration (MIC) andminimumbactericidal or fungicidal concentration (MBC,MFC) were determined for the essential oil. Chemical composition analysis of the essential oil identified a total of 66 compounds. Themain components of essential oilwere Carvone (32%), 1,8-cineole (19%), piperitone (12%), limonene (9%) and limonene (6%) representing 78%of the total oil. Other separated components accounted for less than 22%of the oil. Results of antimicrobial analysis showed that Bacillus cereus (MIC=58 andMBC=232 µg/ml) was more resistance than two other bacterial species. The results of the present study indicated that Mentha longifolia L. essential oil had significant antimicrobial activity; therefore, it can be used as a natural preservation to increase the shelf life of food products.