Abstract The Rhus verniciflua Stokes (RVS) extract is used as a traditional herbal medicine in Southeast Asian countries such as Korea and China. In the present study, one phenolic acid and six flavonoids were isolated from an 80% ethanol RVS extract to examine their antimicrobial activities. These compounds were identified as 3′,4′,7-trihydroxyflavone (1), methyl gallate (2), gallic acid (3), fusti (4), fisetin (5), butin (6), and sulfuretin (7) by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and nuclear magnetic resonance spectroscopy. The antimicrobial activities of compounds 5 and 6 (at a dose of 16 μg/mL each) were superior to that of the control, cycloheximide (at a dose of 25 μg/mL), against Hypocrea nigricans; additionally, the activities of compounds 1 and 2 (at a dose of 8 μg/mL each) were superior to the control against Penicillium oxalicum. Also, chemical compounds 1 and 5 (at a dose of 16 μg/mL each) had higher activities than the control (25 μg/mL) against Trichoderma virens. Chemical compound 1 (at a dose of 8 μg/mL) had a similar activity to that of the control against Bacillus subtilis. The obtained results suggest that the RVS extract could be a promising food and nutraceutical source because of the antimicrobial properties of its phenolic compounds.