The phenylspirodrimane derivatives exhibiting antitumor bioactivities were isolated from the fermentation crude extract of the fungal strain Stachybotrys sp.CPCC 401591.In the study,OSMAC (one strain many compounds) approach was conducted to optimize the fermentation medium.LC-MS/MS-based molecular network analysis was performed for guiding isolation process of secondary metabolites.The routine separation approaches for purification included ethyl acetate extraction,sillica gel column chromatography and preparative HPLC.Eight compounds were obtained and their structures were elucidated as chartarlactam H (1),chartarlactam F (2),stachybotrin (3),chartarlactam K (4),stachybotrylactam (5),F1839-A (6),Mer-VGF724B (7),and 3α-hydroxy-N-isopropyl-carboxyl -phenylspirodrimane (8) by analysis of spectroscopic data including nuclear magnetic resonance,mass spectrometry analysis,and Mo₂(OAc)₄-induced circular dichroism (CD) spectra.The cytotoxic activity of the compounds 1-8 were evaluated by CCK-8 assay in vitro.The results showed that compounds 1-8 displayed moderate inhibition activities on human hepatoma carcinoma cells HepG2,human cervical cancer cells HeLa,and human colon cancer cells HCT116.The result could provide scientific basis for mining more new antitumor compounds from Stachybotrys genus.