Root colonization by antagonistic bacteria is a prerequisite for successful biological control, and the instability of colonization under varying environmental conditions has accentuated the need to improve the colonization activity. Root colonization by Bacillus spp. is mainly determined by chemotaxis and biofilm formation, and both functions are negatively controlled by the global transcription regulator AbrB. Here, we disrupted the gene abrB in Bacillus amyloliquefaciens SQR9, which has been proven to be a promising biocontrol agent of cucumber and watermelon wilt disease. Chemotaxis, biofilm formation, and colonization activities as well as biocontrol efficiency were measured and compared between the wild-type strain of SQR9 and the abrB mutant. The data presented in this article demonstrate that the colonization and biocontrol activity of B. amyloliquefaciens SQR9 could be significantly improved by abrB gene disruption. The results offer a new strategy to enhance the biocontrol efficacy of B. amyloliquefaciens SQR9.