One new cyclopentapeptide, cycloaspeptide H (1), featuring a serine residue, along with seven known compounds (2–8), was isolated from the endophytic fungus Penicillium virgatum GDGJ-227. The planar structure of 1 was elucidated by a comprehensive analysis of NMR and MS spectroscopic spectra, and the absolute configuration was determined by single-crystal X-ray diffraction (Cu Kα) analysis. Compounds 7 and 8 displayed antibacterial activities against Bacillus subtilis and Enterobacter aerogenes with MIC values ranging from 12.5 to 50 μg/mL.