AbstractThe gram‐positive bacterium Bacillus velezensis strain DMW1 produces a high level of antimicrobial metabolites that can suppress the growth of phytopathogens. We investigated the mechanism used by degQ and the degS/degU two‐component system to regulate the biocontrol characteristics of DMW1. When degQ and degU were deleted, the biofilm formation, cell motility, colonization activities, and antifungal abilities of ΔdegQ and ΔdegU were significantly reduced compared to wild‐type DMW1. The expression levels of biofilm‐related genes (epsA, epsB, epsC, and tasA) and swarming‐related genes (swrA and swrB) were all down‐regulated. We also evaluated the impact on secondary metabolites of these two genes. The degQ and degU genes reduced surfactin and macrolactin production and up‐regulated the production of fengycin, iturin, bacillaene, and difficidin metabolites. The reverse transcription‐quantitative PCR results were consistent with these observations. Electrophoretic mobility shift assay and microscale thermophoresis revealed that DegU can bind to the promoter regions of these six antimicrobial metabolite genes and regulate their synthesis. In conclusion, we provided systematic evidence to demonstrate that the degQ and degU genes are important regulators of multicellular behaviour and antimicrobial metabolic processes in B. velezensis DMW1 and suggested novel amenable strains to be used for the industrial production of antimicrobial metabolites.