The aim of this study was to investigate the chemical composition of Origanum vulgare L. essential oil, the inhibitory effect of the oil on the cell viability of Staphylococcus aureus strains isolated from foods, and the influence of sub-inhibitory concentrations of the oil on some physiological attributes of these strains. GC–MS analysis showed that carvacrol (57.71%) was the most prevalent compound in the oil, followed by p-cymene (10.91%), γ-terpinene (7.18%), terpinen-4-ol (6.68%) and thymol (3.83%). The results showed that O. vulgare essential oil at 0.03, 0.6 and 0.12 μL mL −1 inhibited the cell viability of Staph. aureus. At 0.12 μL mL −1 the oil caused cidal effect with decrease ≥3 log cycles of the initial inoculum after 15 min of exposure. Sub-inhibitory concentrations (0.03 and 0.015 μL mL −1) of the oil suppressed some physiological attributes of the Staph. aureus strains such as coagulase, lipase and salt tolerance. The oil interfered on the microbial metabolic activity in a dose-dependent manner. O. vulgare essential oil could be a novel antimicrobial with capability to suppress some physiological characteristics, in addition to inhibit the growth and survival of pathogen bacteria in foods, particularly Staph. aureus.