Aim: To determine uncovered antifungal activity of lichen-derived compound, vulpinic acid, by using chemical-genetic analyses. Methods and Results: Haploinsufficiency and homozygous-profiling assays were performed, revealing that strains lacking GLC7, MET4, RFC2, YAE1 and PRP18 were sensitive to three concentrations (12.5, 25 and 50% of inhibitory concentration) of vulpinic acid and independently validated. To verify inhibition of those genes, cell cycle analysis using flow cytometry was performed and relative expressions were measured. Under vulpinic acid-treated condition, cell cycle was arrested in S and G2/M phases and sensitive strains' relative expressions were significantly lower than the wild type yeast. Conclusions: Vulpinic acid mainly affects cell cycle, glycogen metabolism, transcription and translation to fungi. Significance and Impact of the Study: Although lichen-derived compounds are commercially valuable, few studies have determined their modes of action. This study used a chemogenomic approach to gain insight into the mechanisms of one of well-known lichen-derived compound, vulpinic acid.