Yin Chen Hao Tang (YCHT) is one of the most frequently used prescriptions in the long history of traditional Chinese medicine practice. The prescription contains three Chinese herbs, namely Artemisia capillaries Thunb. (Compositae), Rheum officinale Baillon (Polygonaceae), and Gardenia jasminoids Ellis (Rubiaceae), and has been widely used to treat acute hepatitis with jaundice. In this study, the in vitro anti-HSV-1 and HSV-2 activities of the water extract of YCHT were investigated. Results showed that YCHT water extract inhibited both HSV-1 and HSV-2 infections. However, the inhibition was more effective against HSV-2 than against HSV-1. The IC(50) and IC(90) values of YCHT water extract against HSV-1 infection were in the range of 142.5-150.1 and 191.3-393.9 microg/ml, and against HSV-2 infection they were in the range of 19.6-29.4 and 42.2-97.7 microg/ml, respectively. The water extract of YCHT showed no cytotoxic effect at a concentration of 500 microg/ml or below, and had a CC(50) value of 850.7+/-1.7 microg/ml. The prescription was found to diminish HSV-2 infectivity in a dose-dependent manner, and the activity was influenced by the incubation periods and the incubation temperatures. Concurrent addition of virus with YCHT or pre-treatment of the virus with the prescription extract both protected the cells from infection. In summary, the water extract of YCHT was concluded to inhibit infections by HSV-1 and HSV-2 and this effect was likely mediated through direct inactivation of the virus infectivity.