Background: Zataria 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 Zataria multiflora Boiss. Essential oil on Escherichia coli O157:H7. Methods: Minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of Zataria multiflora Boiss. essential oil (EO) for Escherichia coli O157:H7 at 35°C, the effect of subinhibitory concentrations of EO on growth curve of bacteria up to 24 hours at 25 and 35°C and also production of shigatoxin 2 (Stx2), at 35°C has been determined. Results: MIC and MBC of EO which have been evaluated were 0.04 and 0.06%, respectively the effect of subinhibitory concentrations of EO on bacterial growth curve during 24 hours has been determined by spectrophotometer device at 25 and 35°C and also production of shigatoxin 2 (Stx2), at 35°C has been determined. Subihibitory concentrations of EO, significantly, decreased the production of Stx2 at 35°C in a dose dependent manner. Conclusion: The results showed that Zataria multiflora Boiss. essential oil had inhibitory effect on Escherichia coli O157:H7 and it can be used as a natural preservative in food industry.