Previously, papiliocin was isolated from the swallowtail butterfly Papilio xuthus and its antimicrobial activity was suggested. In this study, the antifungal mechanism of papiliocin was investigated against Candida albicans. Confocal laser scanning microscopy (CLSM) and 1,6-diphenyl-1,3,5-hexatriene (DPH) fluorescence analysis indicated that papiliocin disturbed the fungal plasma membrane. Moreover, the assessment of the release of FITC-dextran (FD) from liposome further demonstrated that the antifungal mechanism of papiliocin could have originated from the pore-forming action and that the radius of the pores was presumed to be anywhere from 2.3 nm and 3.3 nm.