Indole-3-carbinol (I3C) is a naturally occurring constituent of cruciferous vegetables. The aim of this study was to assess the in vitro antimicrobial activity of I3C and its mode of action. By using an NCCLS broth microdilution assay, the activity of I3C was evaluated against human pathogenic microorganisms including clinically isolated antibiotic-resistant bacterial strains. The results indicated that I3C exhibited broad spectrum antimicrobial activities. To elucidate the physiological changes of the fungal cells induced by I3C, we performed a flow cytometric analysis for a cell cycle. The results showed that I3C arrested the cell cycle at the G₂/M phase in Candida albicans. To understand the antifungal mode of action of I3C, the change in the membrane dynamics was monitored by using fluorescence changing experiments against C. albicans. The results suggest that I3C may exert antifungal activity by disrupting the structure of the cell membrane. The present study indicates that I3C has considerable antimicrobial activity, deserving further investigation for clinical applications.