(teleomorph: Ascomycota, Hypocreales, , ) is a destructive fungal pathogen that threatens the production and quality of wheat and barley worldwide. Controlling this toxin-producing pathogen is a significant challenge. In the present study, the commercially available strain (, , , ) FZB42 showed strong activity against The lipopeptide bacillomycin D, produced by FZB42, was shown to contribute to the antifungal activity. Purified bacillomycin D showed strong activity against , and its 50% effective concentration was determined to be approximately 30 μg/ml. Analyses using scanning and transmission electron microscopy revealed that bacillomycin D caused morphological changes in the plasma membranes and cell walls of hyphae and conidia. Fluorescence microscopy combined with different dyes showed that bacillomycin D induced the accumulation of reactive oxygen species and caused cell death in hyphae and conidia. secondary metabolism also responded to bacillomycin D challenge, by increasing the production of deoxynivalenol. Biological control experiments demonstrated that bacillomycin D exerted good control of on corn silks, wheat seedlings, and wheat heads. In response to bacillomycin D, genes involved in scavenging reactive oxygen species were downregulated, whereas genes involved in the synthesis of deoxynivalenol were upregulated. Phosphorylation of MGV1 and HOG1, the mitogen-activated protein kinases of , was increased in response to bacillomycin D. Taken together, these findings reveal the mechanism of the antifungal action of bacillomycin D. Biological control of plant disease caused by is desirable. FZB42 is a representative of the biocontrol bacterial strains. In this work, the lipopeptide bacillomycin D, produced by FZB42, showed strong fungicidal activity against Bacillomycin D caused morphological changes in the plasma membrane and cell wall of , induced accumulation of reactive oxygen species, and ultimately caused cell death in Interestingly, when was challenged with bacillomycin D, the deoxynivalenol production, gene expression, mitogen-activated protein kinase phosphorylation, and pathogenicity of were significantly altered. These findings clarified the mechanisms of the activity of bacillomycin D against and highlighted the potential of FZB42 as a biocontrol agent against .