In the current study we assessed a new crystallized compound, 5-(1-hydroxybutyl)-4-methoxy-3-methyl-2H-pyran-2-one (C-HMMP), from the endophytic fungusColletotrichum acutatumresiding in the medicinal plantAngelica sinensisfor its in vitro antimicrobial, antibiofilm, antioxidant, antimalarial, and anti-proliferative properties. The promising compound was identified as C-HMMP through antimicrobial-guided fraction. The structure of C-HMMP was unambiguously confirmed by 2D NMR and HIRS spectroscopic analysis. Antimicrobial property testing of C-HMMP showed it to be effective against a variety of pathogenic bacteria and fungi with MICs ranging from 3.9 to 31.25 μg/ml. The compound displayed excellent antibiofilm activity againstC. albicans,S. aureus, andK. pneumonia. Furthermore, the antimalarial and radical scavenging activities of C-HMMP were clearly dosede-pendent, with IC50 values of 0.15 and 131.2 μg/ml. The anti-proliferative activity of C-HMMP against the HepG-2, HeLa, and MCF-7 cell lines in vitro was investigated by MTT assay, revealing notable anti-proliferative activity with IC50 values of 114.1, 90, and 133.6 μg/ml, respectively. Moreover, C-HMMP successfully targets topoisomerase I and demonstrated beneficial anti-mutagenicity in the Ames test against the reactive carcinogenic mutagen, 2-aminofluorene (2-AF). Finally, the compound inhibited the activity of α-glucosidase and α-amylase with IC50 values of 144.7 and 118.6 μg/ml, respectively. To the best of our knowledge, the identified compound C-HMMP was obtained for the first time fromC. acutatumofA. sinensis, and this study demonstrated that C-HMMP has relevant biological significance and could provide better therapeutic targets against disease.