Background: Z. multiflora Boiss. is one of the Iranian traditional spices and it has antimicrobial effect on the pathogenic bacteria which are agents for some current foodborne intoxications. Objectives: The present study was conducted to evaluate the antimicrobial activity of Z. multiflora Boiss. Essential oil on Staphylocuccus aureus ATCC 29213. Methods: Minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of Z. multiflora Boiss. essential oil (EO) for Staphylocuccus aureus at 35°C, the effect of subihibitory concentrations of EO on growth of bacteria up to 72 hours and also production of entertoxin E (SEE), all at 25 and 35°C has been determined. Results: MIC and MBC of EO which have been evaluated in 0.005 - 0.06% (V/V) were 0.03 and 0.04%, respectively. Colony counting during 72h showed that 75%MIC of EO in 72h reduced 3 log (cfu/ml) at 25°C and 2, 2 and log (cfu/ml) at 35°C in comparison with control culture. Subihibitory concentrations of EO, significantly, decreased the production of SEE at both 25 and 35°C in a dose dependent manner. Conclusion: The results showed that Z. multiflora Boiss. essential oil had inhibitory effect on Staphylocuccus aureus and it can be used as a natural preservative in food industry.