cBacillus amyloliquefaciens strains are capable of suppressing soilborne pathogens through the secretion of an array of lipopeptides and root colonization, and biofilm formation ability is considered a prerequisite for efficient root colonization. In this study, we report that one of the lipopeptide compounds (bacillomycin D) produced by the rhizosphere strain Bacillus amyloliquefaciens SQR9 not only plays a vital role in the antagonistic activity against Fusarium oxysporum but also affects the expression of the genes involved in biofilm formation. When the bacillomycin D and fengycin synthesis pathways were individually disrupted, mutant SQR9M1, which was deficient in the production of bacillomycin D, only showed minor antagonistic activity against F. oxysporum, but another mutant, SQR9M2, which was deficient in production of fengycin, showed antagonistic activity equivalent to that of the wild-type strain of B. amyloliquefaciens SQR9. The results from in vitro, root in situ, and quantitative reverse transcription-PCR studies demonstrated that bacillomycin D contributes to the establishment of biofilms. Interestingly, the addition of bacillomycin D could significantly increase the expression levels of kinC gene, but KinC activation is not triggered by leaking of potassium. These findings suggest that bacillomycin D contributes not only to biocontrol activity but also to biofilm formation in strain B. amyloliquefaciens SQR9.