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Three new andrastin derivatives, 10-formyl andrastone A (1), 10-demethylated andrastone A (2) and andrastin G (3), together with four known andrastin analogues (4-7) were isolated from an endophytic fungus Penicillium vulpinum. Their structures were determined by 1 D, 2 D NMR, and the absolute configurations were further determined by experimental and calculated ECD spectra. Compound 5 exhibited significant antibacterial activity against Bacillus paratyphosus B with an MIC value of 6.25 µg·mL−1. Compounds 2 and 6 showed remarkable inhibitory activities against Bacillus megaterium with the MIC value of 6.25 µg·mL−1, respectively.