Kombucha fermentation broth has been used as a popular health beverage and an alternative therapy with prophylactic and therapeutic benefit. We tried to establish optimal culture conditions for Kombucha fermentation in milk and to investigate cytotoxicity and antioxidative enzyme activity of Kombucha broth against normal human fibroblasts. The optimal conditions of Kombuch culture were established to 3$0^{circ}C$, 20∼23 hours by DPPH radical scavenging test. There were positive effects on cell growth while no cytotoxicity against primary normal human diploid fibroblasts was found. The activites of glutathione peroxide and catalase in the cells treated by hydrogen peroxide (1 mM) alone and by hydrogen peroxide with Kombucha broth (1 mg/mι) were significantly different (p＜0.05). These results suggest that Kombucha broth could be developed as an antioxidant agent for a new cosmetic material.