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Aim: Validation of antiviral activity of Stephania hernandifolia against HSV-2. Background: Ethnomedicinal plant Stephania hernandifolia, traditionally used for the management of skin, digestive and nerve ailments demonstrated significant anti-HSV-1 activity; similar to Stephania cepharantha having neuroinflammatory and anti-HSV activities. Objectives: Thus, the present study was aimed to validate the potential of the most active fraction-2 (F-2) of S. hernandifolia against HSV-2 in vitro, along with the underlying mode or mechanism of action. Methods: The standardized F-2 was characterized by GC-MS, 1H-NMR, Mass and FTIR spectroscopy. Cytotoxicity (CC50) and antiviral activity (EC50) was evaluated by MTT and Plaque reduction assay. To determine the mode of action, we have used time-of-addition, virus inactivation, and entry (attachment and penetration) assays, followed by semiquantitative PCR. Furthermore, the protein expression levels of immediate early (IE) and early (E) gene products of drug-treated virions were measured by Western blot. Results: The results showed that HSV-2G and ICMR/VU-2012/20, the clinical isolate of HSV-2, were inhibited by F-2 at EC50 of 20.0 and 20.43 µg/ml respectively, with Selectivity Index (SI) of 12. Time-of-addition assay showed that F-2 significantly inhibited HSV-2 infection in Vero cells at 4-8 h post-treatment. The infectivity of the virion was lost within 1h of exposure to F-2 (EC50 and EC99). Furthermore, semi-Q-PCR, and Western blot studies demonstrated significant downregulation of IE and E gene products. The characterization revealed that 2-chloroethyl linoleate is the lead compound in the F-2 fraction. Conclusion: Thus, our results showed that the bioactive fraction F-2, inhibits both IE and E transcription of HSV-2.