Penicillium macrosclerotiorum, was isolated for the first time from the leaves of Ilex pubescens Hook. et Arn (VSN-2022-03-18-001), and found to produce bioactive compounds. Seven compounds were obtained from the fermentation broth and mycelia of Penicillium macrosclerotiorum , and identified as palmitic acid (1), methyl 6-acetyl-5,7,8-trihydroxy-4-methoxy-2naphthoate (2), ergosterol (3), daidzein (4), oleuropein (5), pedunculoside (6), and acteoside (7). Compounds 3, 4, and 7 exhibited antioxidant activity against diphenyl picryl hydrazinyl with IC50 values of 58.06 µg/mL, 2.58 µg/mL, and 12.01 µg/mL respectively. Compounds 3, 5, and 7 showed antibacterial activity against Escherichia coli with MIC values of 0.78 mg/mL, 3.13 mg/mL, and 3.13 mg/mL, while compounds 2, 3, 4, 5, 6, and 7 exhibited inhibitory effect on Candida albicans with MIC values of 0.20 mg/mL, 0.20 mg/mL, 0.39 mg/mL, 0.63 mg/mL, 0.78 mg/mL, and 0.78 mg/mL, respectively. Furthermore, Compound 4 effectively inhibited HepG2 cells, showing an IC50 of 34.03 μg/mL.