Statement of Problems: Herpes Simplex type 1 (HSV-1) is associated with different human infections including oral infection. Acyclovir is used for the treatment of such herpetic infections. However, acyclovir resistant HSVs are increasing nowadays due to the lack of thymidine kinase activity in HSV mutants. So, researchon alternative treatment is urgently required.Purpose: The aim of this study was to examine the effect of essential oil of Zataria multiflora Boiss on herpes simplex type 1 in Vero cell culture.Materials and Method: The essence of Zataria multiflora was prepared by the clevenger apparatus and used for the experiments in Vero cell culture. Cytotoxic effect of increasing concentrations of the essence from 0.001-0.02% was assessed on Vero cell line and antiviral effect of the essence was studied in Vero cells, using plaque reduction method. The data were analyzed by one way ANOVA using SPSS, version 17.Results: The 50% cytotoxic concentration (CC50) of Zataria multiflora essential oil for Vero cells and 50% inhibitory concentration (IC50) were 0.067% and 0.0059%, respectively. Compared to the controls, all the above used concentrations had the inhibitory effect on HSV-1 ( p <0.05). Concentrations of 0.1- 0.2% showed a complete anti-HSV-1 inhibitory effect.Conclusion: The results of this study showed that essential oil of Zataria multiflora Boiss has a significant inhibitory effect on HSV-1, so it may be used as an anti-herpetic mouthwash candidate for the control of oral HSV infections. However, in vivo studies might be necessary for determining its exact toxic effects on human cells. Key words: Zataria multiflora Boiss, Essential oil, Cell culture, HSV-1