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White oyster mushroom (Pleurotus ostreatus Jacq: Fr Kumm) is a food source that contains high nutrition and various secondary metabolites that have pharmacological effects, such as antioxidants and anti-inflammatory. The mushroom is widely used for the prevention of various diseases such as hypertension, hypercholesterolemia and carcinoma. This study assessed the acute toxicity of White Oyster Mushroom ethanol extract, so that the preparation of white oyster mushroom ethanol extract can be increased to become a standardized herbal. The acute toxicity test uses the proposed method, which is a new method recommended for the acute toxicity test of natural substances. Doses tested in this study started from 10 mg / kg body weight to a maximum dose of 5000 mg / kg body weight. Observations of toxicity tests were conducted within the first 24 hours at each stage and the parameters observed were mortality of experimental animals and changes in behavior of experimental animals. The results of the acute toxicity test showed that no experimental animal died during the range of dosing, since the smallest dose reached the maximum dose, other than that there was no visible change in behavior in experimental animals during ethanol extract administration. White oyster mushroom ethanol extract is relatively safe does not show any acute toxicity, because it has a wide dose range.