To develop natural antimicrobial substances from Theaceae, Schima wallichii subsp. liukiuensis was selected from 218 woody plants, and antimicrobial compounds against bacteria, fungi, and yeast were isolated. The antimicrobial activity of ethanol extracts proved higher than those of other organic solvents. The antimicrobial activity of S. liukiuensis extract showed no differences in sesonal variation, but, that of plant part was high in bark at autumn. An antimicrobial substance was isolated from the extract of Schima using column chromatography packed with silica gel and sephadex LH-20, and then a purified antimicrobial substance (Compound I) was obtained using HPLC analysis. The Compound I in the analysis of UV, IR, and GC-MS presumed a triterpene or steroidal saponin, ${alpha}$-sitisterol as aglycon combined three sugars. The minimal inhibitory concentration (MIC) of the Compound I against a bacteria, fungi, and yeast were 1.25 g/L, 5.0 g/L, and 0.040 g/L, respectively. This is much lower than the MIC of hinokitiol, an natural antimicrobial compound used commercially, which suggests that Compound I could be developed as a natural preservative and pharmaceuticals.