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Aims: To study the antimicrobial agents of the Bacillus velezensis strain HC6 and assess the application potential of B. velezensis HC6 in maize. Methods and Results: We applied a dual culture technique to test the antimicrobial activity of B. velezensis HC6 against bacteria and fungi of common contaminated crops. Bacillus velezensis HC6 showed antagonistic action on pathogenic fungi, including Aspergillus and Fusarium, as well as pathogenic bacteria (especially Listeria monocytogenes). When applied in maize, B. velezensis HC6 could also inhibit the growth of multiple pathogenic fungi and reduce their production of aflatoxin and ochratoxin. Three kinds of antimicrobial lipopeptides, including iturin, fengycin and surfactin were identified in B. velezensis HC6 culture supernatant by high-performance liquid chromatography and MALDI-TOF mass spectrometry. Iturin and fengycin showed obvious antimicrobial activity to the tested fungal strains. Conclusions: Bacillus velezensis HC6 produces three kinds of lipopeptides which showed antimicrobial activity against several common pathogenic fungi and bacteria. Bacillus velezensis HC6 is potential to be biocontrol bacteria in maize. Significance and Impact of the Study: Bacillus velezensis HC6 shows obvious antimicrobial activity to important crops pathogenic fungi which usually produce mycotoxins that are harmful to animal and human health. We demonstrate that three different types of lipopeptides produced by B. velezensis contributed to the antimicrobial activity. Bacillus velezensis HC6 has the potential to be effective biocontrol agent in crops.