Introduction Herpes simplex virus type I belongs to herpesviridae. Several studies are ongoing for achieve a drug with good effectiveness and lower side effects, because the incidence of infections caused by this virus is increasing and there is some HSV resistance in the world. In this study, anti-viral effects of rosemary extract against of HSV was evaluated in cell culture. Material and methods: At first the rosemary extract toxicity on Hela cells with trypan blue and MTT methods has been examined and the maximum range of non-toxic concentration on cells has been achieved. At next stages of study direct effect of this concentration of extract examined and after that different concentration of extract in different periods of virus replication have been evaluated. Virus titer with TCID50 method has been measured. Results: Results show, 0.6 mg/ml concentration of rosmary extract is non-toxic on Hela cells and has the maximum effect to prevent HSV-1 replication. The maximum effect of extract has been showed is on immediately after virus adsorption and 1hour after cells infection. In this condition virus titer has decreased compared to other times of virus replication. Conclusion: In accordance with results of this study, herbal basic materials perform their anti-virus effect through interfere with alpha and beta genes expression.