The aims of this study wereto investigate the antifungal activity as a bioactive property of dihydrodehydro-diconiferyl alcohol 9'-O-3-D-glucoside (DDDC9G) and the mode of action(s) involved in its effect. Antifungal susceptibility testing showed that DDDC9G possessed potent antifungal activities toward various fungal strains with almost no hemolytic effect. To understand the antifungal mechanism(s) of DDDC9G, we conducted the following experiments in this study using Candida albicans. Fluorescence experiments using the probe 1,6-eiphenyl-1, 3, 5-hexatriene (DPH) and propidium iodide suggested that DDDC9G perturbed the fungal plasma membrane. Consecutively, the analysis of the transmembrane electrical potential (DeltaPsi) with 3, 3'-dipropylthiadicarbocyanine iodide [DiSC3(5)] and bis-(1,3-dibutylbarbituric acid) trimethine oxonol [DiBAC4(3)] indicated that DDDC9G induced membrane-depolarization. Furthermore, model membrane studies were performed wiith rhodamine-labeled giant unilamellar vesicles (GUVs), calcein encapsulating large unilamellar vesicles (ILUVs), and FITC-dextran (FD) loaded LUVs. These results demonstrated that the antifungal effects of DDDC9G upon the fungal plasma membrane were through the formation of pores with the radii between 0.74 nm and 1.4 nm. Finally, in three dimensional (3D) flow cytometric contour plots, a reduced cell size was observed as a result of osmolarity changes from DDDC9G-induced structural and functional membrane damages.Therefore, the present study suggests that DDDC9G exerts its antifungal effect by damaging the membrane through pore formation in the fungal plasma membrane.