The aim of this study was to evaluate the chemical compositions and antimicrobial activity of Satureja hortensis essential oil against Escherichia coli ATCC 25922, Pseudomonas fluorescens OS8, Alternaria alternata PTCC 5224 and Staphylococcus aureus ATCC 25923 in vitro. In this study, S.hortensis essential oil was purchased from Barij Essence Pharmaceutical Company (Kashan, Iran). The chemical analysis of the essential oil was performed by using Gas chromatography/ mass spectrophotometer (GC/MS). The antimicrobial activity was determined through agar diffusion method (well and disc) and broth micro-dilution based on the CLSI protocol . The results showed that the main compounds in the oil were carvacrol (37.83%) and Isopropyl Myristate (59.14%). The oil showed activity against all tested strains. In all treatments, Gram-negative strains tested were more resistant to the antimicrobial activity of essential oil. Minimum inhibitory concentration (MIC) of S.hortensis essential oil on the Escherichia coli, Pseudomonas fluorescens, Staphylococcus aureus and Alternaria alternata was 2 , 1 , 0.5 and 1( mg/ml) respectively. The agar difiusion tests showed, the reduction of essential oil concentration significantly reduces the diameter of inhibition zone for all tested microorganisms. According to the result of this study and increasing resistance of bacteria to chemical antibiotics, it is recommended that more studies on this plant and antibacterial compounds are needed for treating infections.