Background: About 30% of people in developed countries at least once a year experience a food borne disease. Therefore, there is a need for new methods to prevent the growth of food borne pathogens or decrease the number of them in foods. One of the methods is to use plant essential oils as antimicrobial additives in foods. To establish the usefulness of plant essential oils, they must be evaluated alone or in combination with other preservation factors (such as temperature, storage time and …) first in the laboratory models and then in food model systems. Objective: The aim of this study was to determine of the effect of Zataria multiflora Boiss. essential oil on the growth of Staphylococcus aureus in commercial barley soup using different essential oil concentrations (0.0, 0.005, 0.015 and 0.03%) and temperatures (8 and 25˚C) during 21 days. Results: The effect of different concentrations of essential oil on growth rate of Staphylococcus aureus was statistically (ANOVA) significant (p<0.01). The correlation coefficient of Zataria multiflora Boiss. essential oil with log10 of the number of Staphylococcus aureus was -0.588. The effect of storage time on growth rate of the organism was also statistically (ANOVA) significant (p<0.05). The correlation coefficient of storage time with log10of the number of microorganism used in this study was -0.2 which showed a decrease in the growth rate of the organism when the storage time was increased. In addition the correlation coefficient of storage temperature with log10 of the number of Staphylococcus aureus was 0.449. In other words, by decreasing the storage temperature (from 25 to 8˚C), the decreasing of the growth rate of the microorganism was observed. The effect of different storage temperatures and their interaction with different concentrations of essential oil on growth rate of Staphylococcus aureus was also statistically (ANOVA) significant (p<0.01). Conclusion: The results suggested that the Zataria multiflora Boiss. essential oil can be considered as a natural preservative in some foods.