Niu-Chang-Chih, which is the fruiting body of Antrodia camphorata, is a folk medicine that has traditionally been used in Taiwan for treating food and drug poisoning, liver disease, and cancer. By using HBV producing cell line as an in vitro model system, we found that Niu-Chang-Chih displays anti-HBV effects on both wild-type and lamivudine-resistant mutants. The active compounds responsible for the anti-viral activities of Niu-Chang-Chih were further investigated and identified by anti-HBV assay directed fractionation. Specifically, out of the ten pure compounds isolated from Niu-Chang-Chih which includes one biphenyl, four ergostanes, and five lanostanes derivatives, 2,2',5,5'-tetramethoxy-3,4,3',4'-bis(methlylene-dioxy)-6,6'-dimethyl-biphenyl was positively identified as the single active compound responsible for the anti-viral effects on both wild-type and lamivudine-resistant mutant HBV.