Herpes is a challenging opportunistic infection caused by herpes simplex virus 1 (HSV-1). Emergence of drug resistance is the worst catastrophe that encountered the dedicated efforts to eradicate the disease. This urged the scientists to search for other alternatives from the natural products sanctuary. In this study, aqueous, methanol and ethanol extracts of Orthosiphon stamineus leaves (ALE, MLE & ELE) were prepared and their yield extract and phytochemical contents were determined. Their anti HSV-1 effect was screened using plaque reduction assay and their safety limit was assessed after determining their cytotoxicity against Vero cells using MTT assay. This was followed by mechanism elucidation study which included pre-treatment, attachment, virucidal, penetration, virus yield and time removal and time addition assays. MLE got the highest yield extract and phytochemical content as compared to the others. All of them were considered as non-toxic to Vero cells as their (CC_50s > 0.2 mg/ml). Furthermore, their anti-HSV-1 effect shows that all of them had a selective effect in the following order (MLE>ELE>ALE. Their anti-HSV-1 effect was ascribed to their direct virucidal effect or interference with the viral entry through modifying the viral ligand molecules or preventing fusion of the viral envelop with the host cell membrane. In this regard, MLE produced the most powerful effect and it was the only which inhibited the intracellular activity of the virus at all the replication stages. Overall, O. stamineus leaves can be used to develop new antiherpetic agent.

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