Thuja orientalis is used as a herbal medicine in Korea and China. This study conferred the antimicrobial activity of wild and planted Thuja orientalis. The antimicrobial activity was evaluated by disc diffusion and minimal inhibition concentration (MIC) method against eight strains. The ethyl acetate fraction of wild and planted T. orientalis leaves showed more antimicrobial activity as compared to the water fraction. The ethyl acetate fraction of wild T. orientalis revealed stronger antimicrobial activity than the fraction of planted T. orientalis, while the antimicrobial activity of the water fraction did not show the difference between wild and planted T. orientalis. The present study showed that there was little difference of antimicrobial activity between wild and planted T. orientalis.