The crude extracts of Engleromyces goetzei P. Hennings were obtained by leaching,centrifugation,precipitation and lyophilization. The crude polypeptides were obtained after extracting and isolating the crude extracts with G25 gel chromatography. The antibacterial activities were measured with paper filtering method and plate confrontation method on the diameter of inhibition zone as an indicator. It inhibited all types of tested agricultural pathogenic fungi on mycelium growth.Among them,the antibacterial activity against Gibberella zeae,Drupe anthrax and Verticilllium albo atrum was highest,with the IC50 of 8.70 μg/mL,9.48 μg/mL and 8.96 μg/mL,respectively. The crude polypeptides were further purified with HPLC to obtain an electrophoretically pure antimicrobial peptide. It had wide antimicrobial activities and was worth for the further studying.