Background: It was shown that olive leave extract has antifungal, antibacterial and antiviral activities. The effects of OLE on herpes simplex virus-1 (HSV-1) have not been systematically investigated yet. The aim of this study was to examine the in vitro effect of olive leaf hydroalcoholic extract (OLHE) on HSV-1. Methods: Virucidal effect and viral replication in Vero cell line were studied in the presence of various concentrations of OLHE applied at different time intervals using a standard plaque assay method. The 50% cytotoxic concentration (CC50), 50% inhibitory concentration (IC50), and therapeutic index of OLHE were determined. Results: OLHE showed virucidal effect on HSV-1 in concentrations >1 mg/mL. The CC50 of OLHE for Vero cells and IC50 were 1.75 and 0.66 mg/mL, respectively. When applied to cell culture infected with the HSV-1, one hour earlier, OLHE showed no antiviral activities. When applied to the cells followed by the virus infection one hour later, or to the media containing the virus and the combination was added to cell culture one hour later, OLHE showed anti-HSV-1 activities at concentrations >1 mg/mL. Conclusion: OLHE has anti-HSV-1 activity likely due to the prevention of virus entry into the cells.