Furaneol, a key aroma compound found in strawberry, pineapple, and processed foodstuffs, has been known to possess various biological activities on animal models. In this study, the antimicrobial effects of furaneol against human pathogenic microorganisms were investigated. The results indicated that furaneol displayed a broad spectrum of antimicrobial activities against Gram-positive and Gram-negative bacteria and fungi without hemolytic activity on human erythrocyte cells. To confirm the antifungal activity of furaneol, we examined the accumulation of intracellular trehalose as a stress response marker on toxic agents and its effect on dimorphic transition of Candida albicans. The results demonstrated that furaneol induced significant accumulation of intracellular trehalose and exerted its antifungal effect by disrupting serum-induced mycelial forms. These results suggest that furaneol could be a therapeutic agent having a broad spectrum of antimicrobial activity on human pathogenic microorganisms.