The possibility of producing natural antioxidants from mycelium-free culture broth of Pleurotus ostreatus was investigated. The mycelium-free liquid media from which the mycelium was removed after 20 days of culture showed high extraction yield an ethanol extraction proceeded by concentration. The antioxidative activity was observed from the ethanol extracts of mycelium-free culture broth of Pleurotus ostreatus. This is a strong evidence of the extracellular antioxidative substances from the liquid cultured mycelium. The antioxidative activity was more significant in the oxidation experiment at 100℃ compared to that at 60℃. This is a possible suggestion that the antioxidative substances would be a heatstable components.