In a previous study, we investigated the antioxidative and cellular protective effects of Parthenocissus tricuspidata stem extracts. In this study, we prepared nano-emulsion containing P. tricuspidata stem extract to improve skin permeation. The particle size of the nano-emulsion using the microfluidizer was 302 nm. Its loading efficiency was over 86%. The size distribution of the nano-emulsion took a monodispersed form and the nano-emulsion was more stable than typical emulsion without using microfluidizer during a 2 week period. In vitro skin permeation study of nano-emulsion containing P. tricuspidata stem extracts was carried out using Franz diffusion cell. The 1,3-butylene glycol used as a control group had 32.59% skin permeation efficiency. The skin permeation efficiency of the nano-emulsion was 42.47%. Also, we observed the antibacterial activity of the ethyl acetate fraction on skin flora for prospective applications as a natural antimicrobial. The ethyl acetate fraction had antibacterial activities higher than methyl paraben on Staphylococcus aureus, and Bacillus subtilis. These results indicate that nanoemulsion containing P. tricuspidata stem extracts could possess valued applications in cosmetic formulations for improving skin permeation. Also, based on the antibacterial activities on skin flora, antioxidative and cellular protective effects shown in our previous study, we suggest that P. tricuspidata stem extracts could be used as functional cosmetic materials.