BACKGROUND: Development of new and effective therapeutics for sexually transmitted herpes simplex virus-2 (HSV-2) infection is important from public health perspective. With an aim to identify natural products from medicinal plants, in the present study, the potential of Terminalia chebula Retz was investigated for its activity against HSV-2. METHODS: Fruits of Terminalia chebula Retz were used to prepare 50% ethanolic extract. In addition, chebulagic acid and chebulinic acid both purified from T. chebula were also used. The extract as well as purified compounds were first used to determine their in vitro cytotoxicity on Vero cells by MTT assay. T. chebula extract, chebulagic acid, chebulinic acid along with acyclovir were subsequently assessed for direct anti-viral activity, and their ability to inhibit attachment and penetration of HSV-2 to the Vero cells. In addition, their anti-HSV-2 activity was also determined by in vitro post-infection plaque reduction assay. RESULTS: Cytotoxicity assay using Vero cells revealed CC50?=?409.71?±?47.70 ?g/ml for the extract whereas chebulagic acid and chebulinic acid showed more than 95% cell viability up to 200 ?g/ml. The extract from T. chebula (IC50?=?0.01?±?0.0002 ?g/ml), chebulagic (IC50?=?1.41?±?0.51 ?g/ml) and chebulinic acids (IC50?=?0.06?±?0.002 ?g/ml) showed dose dependent potent in vitro direct anti-viral activity against HSV-2. These also effectively prevented the attachment as well as penetration of the HSV-2 to Vero cells. In comparison, acyclovir showed poor direct anti-viral activity and failed to significantly (p?>?0.05) prevent the attachment as well as penetration of HSV-2 to Vero cells when tested upto 50 ?g/ml. However, in post-infection plaque reduction assay, T. chebula extract, chebulagic and chebulinic acids showed IC50 values of 50.06?±?6.12, 31.84?±?2.64, and 8.69?±?2.09 ?g/ml, respectively, which were much lower than acyclovir (71.80?±?19.95 ng/ml). CONCLUSIONS: The results presented herein suggest that T. chebula extract, chebulagic and chebulinic acids have higher direct antiviral activity against HSV-2 and efficacy to inhibit virus attachment and penetration to the host cells as compared to acyclovir. However, acyclovir is more potent to inhibit post-infection virus replication. Hence, T. chebula may be a useful candidate for developing alternative therapy for prevention of sexually transmitted HSV-2 infection. ?.