Background: Long Dan Xie Gan Tan (LDXGT), a decoction of radix gentianae for purging the pathogenic inflammation of the liver, is a widely used prescription among many in traditional Chinese medicine. The prescription is primarily used to treat the disorders induced by damp-heat in the liver and the gall bladder. Methods: In this study, the in vitro anti-herpes simplex virus type-1 (HSV-1) and type-2 (HSV-2) activity of the water extract of LDXGT was investigated. Results: LDXGT water extract was shown to exhibit anti-HSV activity. The IC50 values of LDXGT against HSV-1 and HSV-2 infections were 257.5 ± 12.2 and 494.6 ± 1.8 µg/ml, respectively. It had a CC50 value of 4,077.2 ± 2.4 µg/ml towards Vero cells and showed no cytotoxic effect at a concentration of 2,000 µg/ml or below. The prescription was also found to inactivate HSV-2 infectivity in a dose-, time- and temperature-dependent manner. Conclusions: In summary, the water extract of LDXGT was concluded to inhibit HSV-1 and HSV-2 infection at different magnitudes of potency, and our observations also suggested that the effect was likely mediated by directly inactivating the virus infectivity.