Ganoderma leucocontextum is a well‐known traditional medicine in Tibet Autonomous Region, which has benefits, such as anti‐hypoxia, neurotrophic action on nerves, easing coughs and relieving asthma, strengthening the body and prolonging life. However, few research have focused on its negative effects, possibly jeopardizing its safety. The purpose of this study is to evaluate the acute and subacute toxicity of an alcoholic extract from G. leucocontextum (GLA) in vivo. The phytochemical characterization analysis showed that alcoholic extract from G. leucocontextum were rich in polysaccharides, triterpenoids. Then, in acute oral toxicity, male and female mice from Institute of Cancer Research (ICR) were orally administered with 16 g/kg GLA and were observed for 14 days. In the subacute toxicity, male and female Sprague–Dawley (SD) rats were orally administered with 2, 4, and 8 g/kg doses of GLA for 28 days. There was no death or clinical changes in male and female mice in the acute toxicity test. During the subacute toxicity test, the difference in body weights, food consumption, biochemical and hematological parameters, and organ coefficients between treated and control groups were unrelated to GLA treatment. The obtained data show that the GLA had no significant toxic effects when administered orally to male and female rats in acute and subacute toxicity.