Isoquercitrin is a flavonoid isolated from Aster yomena, which has been used as a traditional medicinal herb. In the present study, we investigated the antifungal activity and the underlying mechanism of isoquercitrin. Isoquercitrin had a potent effect in the susceptibility test against pathogenic fungi and almost no hemolysis. Propidium iodide and potassium release assays were conducted in Candida albicans, and these studies confirmed that isoquercitrin induced membrane damage, thereby, increasing permeability. Membrane potential was analyzed using 3,3'-dipropylthiacarbocyanine iodide [DiSC3(5)], and the transition of membrane potential was indicated by an increased fluorescence intensity. To further analyze these results using model membranes, giant unilamellar vesicles and large unilamellar vesicles that encapsulated calcein were prepared and the detection of calcein leakage from liposomes indicated that membrane was disturbed. We further verified membrane disturbance by observing the disordered status of the lipid bilayer with 1,6-diphenyl-1,3,5-hexatriene fluorescence. Moreover, changes in size and granularity of the cell were revealed in flow cytometric analysis. All these results suggested the membrane disturbance and the degree of disturbance was estimated to be within a range of 2.3 nm to 3.3 nm by fluorescein isothiocyanate-dextran analysis. Taken together, isoquercitrin exerts its fungicidal effect by disturbing the membrane of cells.