This study was conducted to evaluate the contents of total polyphenol and flavonoid, and the effect of antioxidant, antimicrobial activities and cytotoxicity in vitro by different solvent fractions from Orostachys japonicus. The ethylacetate fraction extract for O. japonicus contained 634.48 ㎍/g polyphenol and 205.20 ㎍/g flavonoid. The ABTS radical scavenging ability of ethylacetate fraction extract at 1 ㎎/㎖ was higher than 95% which is comparable to ascorbic acid of 97%. The APX enzymatic activity and CAT activity were 1125.89 μmol ascorbate oxidized/min/㎎ protein and 119.87 H2O2 decomposed/ min/㎎ protein, respectively. In disc agar plate diffusion assay, the extract gave rise to a larger inhibition circle with Listeria monocytogenes, Staphylococcus epidermidis, Staphylococcus aureus and Malassezia furfur strains compared with antibiotics kanamycin suggestive of high antibiotic activity. The cytotoxicity of extracts of O. japonicus was significant differences between solvent fractions. That is, the cytotoxic effect against human cancer cell was higher in ethylacetate fraction extract than other fraction extracts. These results suggest that fraction extract of O. japonicus might be very effective and economical in developing natural antioxidant and antimicrobial.