Background: Aspergillus flavus is a toxic contaminant in foods, which can induce mutagenic, teratogenic, and carcinogenic effects. In last decades, synthetic fungicides have been used for inhibition of fungal growth in plants. The public attention was also attracted to contamination of food chain by these chemicals. Therefore, in the current study, we decided to use Zataria multiflora (ZM) essential oil to inhibit the Aspergillus flavus growth. Method: The essential oil from ZM was obtained by hydro-distillation and analyzed by GC/MS. The minimal inhibitory concentration (MIC) and minimal fungicidal concentration (MFC) of ZM essential oil were determined at different concentrations (0-1000 ppm). Results: In this study, Carvacrol (33.45%) and Thymol (34.44%) were the most frequent compounds of the ZM essential oil. The minimum inhibitory and fungicidal concentrations were 100 and 400 ppm, respectively. Conclusions: The presence of phenolic compounds such as Thymol and Carvacrol, as the major components of ZM essential oil inhibits the Aspergillus flavus growth. Furthermore, this essential oil has a very strong anti-bacterial effect. Considering these results, it seems that ZM essential oil is potentially an appropriate natural alternative for controlling Aspergillus flavus fungus.