Ethyl acetate extract of endophytic fungus from Penicillium sp. of kunyit putih showed antibacterial activity in vivo but no acute toxicity. However, the extract may have toxic effects on major organs for long-term consumption. This study was carried out in order to test sub-chronic toxicity of the ethyl acetate from endophytic fungus Penicillium sp. of kunyit putih against mice (Mus musculus). A total of 50 male mice were divided into five groups. Groups I, II, III and IV were orally administered with ethyl acetate extracts of 250, 500, 1000 and 2000 mg/kg body weight (BW), respectively. Group V was used as a control without extract treatment. A toxic symptom was observed by analyzing several parameters, namely change in BW, hematologic and biochemical properties (SGOT & SGPT), macroscopic organs, and relative organ weight. The results showed that there was toxic symptom and statistically significant difference in the parameters of SGOT, SGPT and heart weight between treated and control groups. Based on analysis, we concluded that ethyl acetate extract from endophytic fungus Penicillium sp. of kunyit putih had subchronic toxicity effect.