We analyzed the biological activity and composition of total triterpenoids extracted from the liquid fermentation culture of the medicinal mushroom Sanghuangporus sanghuang. S. sanghuang total triterpenoid extract (STTE) had the strongest inhibitory effect on Staphylococcus aureus, and STTE at 2-fold the minimum inhibitory concentration had a significant effect on the growth of four types of bacteria and caused marked nucleic acid leakage. Scanning electron microscopy confirmed that STTE destroyed bacterial cell walls. STTE also significantly inhibited the proliferation of Caco-2 cells and disrupted their morphology. Flow cytometry revealed that STTE induced cell cycle arrest and induced early and late apoptosis of Caco-2 cells. Infrared spectroscopy, and gas chromatography-mass spectrometry resulted in the detection of a variety of triterpenoids (e.g., pentacyclic triterpenoid, lanolinane triterpenoid, and trans-squalene) as well as other small molecules (e.g., esters, acids, and aldehydes). Our findings indicate that STTE has antibacterial and antitumor activity and will enable screening of novel fungal-derived anticancer and antibacterial compounds.