This study was carried out to investigate the antimicrobial activities of organic extracts obtained from the fruit of Thuja orientalis L. The native fruits in Korea were collected and extracted by 80% ethanol, and the extract was sequentially fractionated with n-hexane, chloroform, ethylacetate, and butanol. The fraction yields of n-hexane, chloroform, ethylacetate, butanol and water of ethanol extract were 10.15%, 10.05%, 1.45%, 45.35% and 27.55%, respectively. n-Hexane-soluble fraction showed the highest antibacterial activity against gram positive bacteria, while the chloroform, ethylacetate, butanol and aqueous fractions did not show any antibacterial activity. Minimum inhibitory concentration (MICs) on Staphyloycoccus aureus, Bacillus subtilis and Sateptococcus pneumoniae, n-hexane-soluble fraction were $100;{mu}g$, $500;{mu}g$ and $50;{mu}g$/disc, respectively. The antibacterial activity was not destroyed by heating at 80, 100, $120^{circ}C$ for 30 min and was not affected by pH. In the inhibitory test against the Staphylococcus aureus, Bacillus subtilis and Sateptococcus pneumoniae, n-hexane-soluble fraction showed potent growth inhibition at the concentration of 0.1 and $0.5;{mu}g/mL$ for 12~24 hours and n-hexane-soluble fraction did not show any mutagenic activity.