Chemical investigation of the plant endophytic fungus Pestalotiopsis sp. afforded two new butenolides, pestalolides B and C (1 and 2), two new diphenyl ethers, pestalotethers E and F (3 and 4), together with seven known compounds (5–11). Their structures were established by extensive spectroscopic analyses and the absolute configuration of 1 was further confirmed by ECD calculation. Structurally, compound 1 represents the first example of a new type of butenolide derivatives, bearing an unprecedented carbon scaffold having 13 carbon atoms. Compounds 1–4 were evaluated for the cytotoxic activities, and compound 1 showed remarkable inhibitory potency against human tumor HL60, U87MG, MDA-MB-231, and HEP-3B cell lines, with IC50 values of 1.42, 5.90, 5.90, and 4.29 μM, respectively.