In this study, the antifungal activity and mode of action(s) of hibicuslide C derived from Abutilon theophrasti were investigated. Antifungal susceptibility testing showed that hibicuslide C possessed potent activities toward various fungal strains and less hemolytic activity than amphotericin B. To understand the antifungal mechanism(s) of hibicuslide C in Candida albicans, flow cytometric analysis with propidium iodide was done. The results showed that hibicuslide C perturbed the plasma membrane of the C. albicans. The analysis of the transmembrane electrical potential with 3,3'-dipropylthiacarbocyanine iodide [DiSC3(5)] indicated that hibicuslide C induced membrane depolarization. Furthermore, model membrane studies were performed with calcein encapsulating large unilamellar vesicles (LUVs) and FITC-dextran (FD) loaded LUVs. These results demonstrated that the antifungal effects of hibicuslide C on the fungal plasma membrane were through the formation of pores with radii between 2.3 nm and 3.3 nm. Finally, in three dimensional flow cytometric contour plots, a reduced cell sizes by the pore-forming action of hibicuslide C were observed. Therefore, the present study suggests that hibicuslide C exerts its antifungal effect by membrane-active mechanism.