Background: The growing interest in substitution of chemical food preservative with natural ones has fostered researches on plant essential oils and extracts. Objectives: The purpose of the current study was to evaluate the effect of Zataria multiflora Boiss. essential oil on growth respose, the percent of growth inhibitory and morphology of Penicellium citrinum. Methods: Different concentrations of the essential oil (0, 50, 100, 200, 400, 600 ad 1000 ppm) were used in agar dilution method to evaluate growth and spore production parameters. The cultured mold were studied by scanning electron microscope. Values among groups were compared using 1-way ANOVA. Results: It was found that the effect of different concentrations of essential oil on radial growth and sporulation was statistically significant (p<0.05). The minimum inhibitory concentration (MIC) and minimum fungicidal concentration (MFC) both were 400ppm. According to scaning electron microscopy the treatment with the oil led to alterations in hyphal morphology. Conclusions: Our results suggest that Zataria multiflora Boiss. essential oil can be used as a natural preservative against Pencillium citrinum in foods.