Based on bioactivity-oriented isolation, the EtOAc extract of a culture broth of the endophytic fungus Perenniporia tephropora Z41 from Taxus chinensis var. mairei, with strong anti-Pyricularia oryzae activity, afforded a new sesquiterpenoid, perenniporin A (1), together with three known compounds, ergosterol (2), rel-(+)-(2aR,5R,5aR,8S,8aS,8bR)-decahydro-2,2,5,8-tetramethyl-2H-naphtho[1,8-bc]genfuran-5-ol (3), and albicanol (4). Their structures were elucidated by means of spectroscopic methods. All the isolated compounds and the EtOAc extract of P. tephropora Z41 (EPT) were evaluated for their cytotoxic activity against three human cancer cell lines (HeLa, SMMC-7721, and PANC-1). EPT demonstrated significant cytotoxicity with IC(50) values ranging from 2 to 15 μg/mL. Compound 2 was the most cytotoxic constituent against the tested cell lines with IC(50) values of 1.16, 11.63, and 11.80 μg/mL, respectively, while compounds 1, 3, and 4 exhibited moderate cytotoxicity with IC(50) values ranging from 6 to 58 μg/mL. We conclude that the endophytic fungus P. tephropora is a promising source of novel and cytotoxic metabolites.