To characterize the secondary metabolites and evaluate their bioactivities of Penicillium glabrum SF-61290, the study was carried out to isolate and purify the ethyl acetate extract of its fermentation broth using silica gel column chromatography and preparative HPLC, and identify the structures of the compounds by modern spectroscopic methods such as NMR, MS, and so on. Additionally, the in vitro cytotoxic and antibacterial activities of these compounds were assessed using the MTT assay and the microdilution method, respectively. Ten compounds were isolated from the ethyl acetate extract of the strain fermentation broth, and identified as penicilazaphilone O (1), sclerotiorin (2), isochromophilone XV (3), 8a-epi-hypocrellone A (4), arvoredol (5), geumsanol G (6), geumsanol D (7), penicilazaphilone B (8), geumsanol B (9), and isochromophilone VI (10). Among these, compound 1 is a new azaphilone derivative. Compounds 3, 5, 7, and 10 exhibited significant cytotoxicity against the colorectal cancer HCT-8 cell line, with IC₅₀ values of 13.1, 27.84, 17.96, and 54.17 μmol/L, respectively, outperforming the positive control 5-fluorouracil (IC₅₀ = 61.17 μmol/L). Compounds 1-10 exhibited no obvious activities against Escherichia coli, Staphylococcus aureus, and Streptococcus suis, with minimal inhibitory concentration all exceeding 100 μg/mL.