Powdery mildew disease of cucurbits is caused mainly by Podosphaera fusca, which is one of the most important limiting factors in cucurbit production worldwide. Previously we reported that Bacillus amyloliquefaciens biocontrol strain SD-32 produces C17 bacillomycin D and [Ile 2002]surfactin, and that these metabolites play important roles in SD-32's biocontrol over cucumber gray mold disease. Our further investigation demonstrated that the culture broth and its supernatant suppressed cucumber powdery mildew disease in greenhouse experiments. However, the active principle(s) remained unknown. The active compound was isolated from the culture supernatant after anti-powdery mildew disease activity-guided purification and identified as prumycin. Prumycin significantly suppressed the disease, whereas bacillomycin D and [Ile 2002]surfactin did not. Prumycin did not induce the expression of plant defense genes (PR1a and VSP1), suggesting that it does not act via plant defense response. Light microscopic observations of prumycin-treated cucumber cotyledon suggested that prumycin inhibits the conidial germination of P. fusca. This study demonstrates that prumycin is a major factor in SD-32's suppression of cucumber powdery mildew disease. Our findings shed light for the first time on prumycin's role in biocontrol by Bacillus against this disease. © 2017 Society of Chemical Industry.