Insect pest control has been a major problem faced by farmers around the world and there has been no perfect solution to handle it. The use of insecticides derived from chemically active compounds has made negative impacts such as the emergence of resistant pest and disruption of ecosystems. In addition, it raised many other negative impacts on the environment and on non-target organisms. Therefore, an alternative is needed to replace chemical insecticides. Entomopathogenic fungus is one of the potential biological control organism to suppress the growth of insect pests. This study aims to extract mycotoxin from entomopathogenic fungi isolated from the apple orchard at Batu, East Java, Indonesia and determine the level of toxicity against Tenebrio molitor larvae. Mycotoxin was extracted using precipitation with ammonium sulphate and dialysis. It was subjected to molecular weight analysis and toxicity test to determine the LC50 value. Results showed that protein bands were found between 20 kDa to 55 kDa. Based on the study, the mycotoxin showed a LC50 at a concentration of 42.78 μg · mL −1 .