This study was designed to evaluate the chemical constituents of Zataria multiflora Boiss. essential oil (EO) and its inhibitory effect on enterotoxin type C production by Staphylococcus aureus ATCC 6538. Moreover, the effect of this EO on gene expression of staphylococcal enterotoxin type C (SEC) was assessed. The effect of different concentrations of EO on enterotoxin C production was evaluated using 3-enzyme-linked immunosorbent assay. The reducing effect of EO subinhibitory concentrations on gene expression of enterotoxin was assayed using real-time polymerase chain reaction as well. EO at the level of 0.005% had no inhibitory effect on enterotoxin C production, and by increasing the subinhibitory concentration of the EO to 0.015%, an inhibitory effect was observed. Comparing the relative gene expression of SEC in control and treatment groups showed that the EO concentrations of 0.0075 and 0.015% had a significant downregulation (10.7 and 23.8 times, respectively) of sec gene.