Aims: Phytochemical analysis showed Orthosiphon stamineus (OS) possessed bioactive compounds with antiviral properties against Herpes Simplex Virus Type 1 (HSV-1). However, there isn’t any study reported so far on OS virucidal properties towards HSV-1. Thus, this study aims to investigate virucidal mechanism of OS aqueous extract that possibly acts as a potent entry inhibitor against HSV-1 infection. Methodology and results: Virucidal attachment and penetration assays were done via plaque assay to investigate the virucidal anti-HSV-1 mechanism of OS. The aqueous extract of OS leaves (OSA) was found to reduce HSV-1 plaques in virucidal assays. Inhibitory effect by OSA was observed as early as 30 min after exposing OSA to HSV-1 in a concentration-dependent manner suggesting a direct anti-HSV-1 property of OSA. Further investigation of the stages in which OSA inhibits HSV-1 shows virions treated with OSA failed to attach onto the host cell which implicated a role of OSA in blocking HSV-1 attachment to its host. OSA was also found to reduce HSV-1 plaques in penetration assay. Further evaluation using transmission electron microscopy (TEM) on OSA treated virion showed defective HSV-1 virion without envelope and the remaining capsid was altered. Conclusion, significance and impact of study These findings concluded that Orthosiphon stamineus leaves extract have virucidal activity by disintegrating HSV-1 virion structure and interfering with the attachment and penetration of the virus into the host cell. Thus, through the new mechanism against HSV-1, OS has the potential to be further developed as an anti-HSV-1 agent.