Staphylococcus aureus is recognized as one of the major cause of infections in communities and hospitals. In this study, the chemical composition and antistaphylococcal activity of Zataria multiflora Boiss essential oil were evaluated, along with its synergistic activity with vancomycin. The essential oil was obtained by hydrodistillation from aerial parts of the plant and analyzed using GC and GC-MS. Thymol (38.7%), carvacrol (15.3%) and rho-cymene (10.2%) were the main components of the oil. Antibacterial activity of the oil was evaluated by microbroth dilution assay against clinical isolates of methicillin -resistant (MR) and methicillin-sensitive (MS) strains of S. aureus. The oil showed high antibacterial activity against MRSA and MSSA with minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) values in the range of 0.25-1 and 0.5-2microl/ml, respectively. The synergistic action was evaluated by microtiter assay. The essential oil exhibited synergism with vancomycin and displayed the ability to enhance the activity of vancomycin. This study showed that Z. multiflora oil has significant antimicrobial activity against clinical isolates of S. aureus, especially MRSA, and might be useful in controlling MRSA infections.