Bacillus amyloliquefaciens, as a biocontrol bacterium, mainly produces secondary metabolites to resist pathogenic microorganisms. In the study, B. amyloliquefaciens fmbJ could produce several antimicrobial lipopeptides (bacillomycin D, surfactin, and fengycin). In order to clarify the influence of transcriptional regulatory genes (codY, comA, degU, and spo0A) regulating the biosynthesis of lipopeptides, especially bacillomycin D, these genes in fmbJ were knocked out. The results showed that the productions of bacillomycin D were significantly reduced compared with that of fmbJ. The changes of lipopetides production in fmbJ with the genes deleted were then analyzed by comparative transcriptomics. Their deletion induced great changes in the levels of transcripts specifying metabolic pathways, quorum sensing system and substance transport system in fmbJ. Moreover, overexpression of these genes improved the productions of bacillomycin D. In particular, the overexpression of spo0A enhanced bacillomycin D yield up to 648.9 ± 60.9 mg/L from 277.3 ± 30.5 mg/L. On the contrary, the yields of surfactin in fmbJΔcodY and fmbJΔdegU were significantly improved. And, the regulatory factor CodY had no significant effect on the synthesis of fengycin. In addition, it was found that CodY had a concentration dependence on bacillomycin D synthesis. This study indicated the direction of genetic manipulation to improve the yields of antimicrobial lipopeptides and laid a theoretical foundation for the industrial production of lipopeptides.