An endophytic fungus was isolated from Erythronium japonicum Decne. Using a tissue separation method, and this fungus was identified by 28S rDNA sequencing and phylogenetic analysis. The strains were cultured in potato dextrose (PD) and yeast and malt extract (YM) media, and their secondary metabolites were analyzed using high-performance liquid chromatography (HPLC) and mass spectrometry (MS). This strain was cultivated on a large scale in YM medium, while the secondary metabolites were separated and purified using silica gel chromatography. The cytotoxicity of the compounds isolated from secondary metabolites was determined by MTT assay. The endophytic fungus from Erythronium japonicum Decne. was identified as Eupenicillium sp. NF2_PD013. HPLC and MS analyses indicated that there were more secondary metabolites of this endophytic fungus in YM medium with a higher yield than those in PD medium. The four chlorine-containing compounds obtained from the secondary metabolites in YM medium were identified as geodin, dihydrogeodin, 3’-chlorotrypacidin, and monochlorosulochrin. Geodin and 3’-chlorotrypacidin showed significant inhibitory effects on the proliferation of breast cancer cells MCF-7 and hepatocellular carcinoma cells HepG2.