Abstract The essential oil of the aerial parts of Zataria multiflora from Jandagh area in Iran was prepared by hydrodistillation and its chemical constituents were analyzed by a combination of capillary GC and GC-MS. Twenty five components were identified of which, carvacrol (50.6 %), thymol (13.4 %) and pcymene (8.3 %) were the main constituents. The antibacterial activity of the oil was determined against 9 clinical isolates of Staphylococcus aureus and 6 Gram positive and Gram negative bacterial standards by disc diffusion. Minimum inhibitory and antibacterial concentrations (MIC and MBC) were determined by broth the microdilution assay. Considerable inhibitory activity was shown against all test bacteria shown by disc diffusion (except P. aeruginosa) and MIC and MBC values were within the range of 0.66 - 2.64 mg/ml for the susceptible organisms.