Bacillus velezensis FZB42 is a prominent plant growth-promoting rhizobacterium and biocontrol agent known for producing a wide array of antimicrobial compounds. The capability to genetically manipulate this strain would facilitate understanding its metabolism and enhancing its sustainable agriculture applications. In this study, we report the first successful implementation of high-efficiency CRISPR-Cas9 genome editing in B. velezensis FZB42, enabling targeted genetic modifications to gain insights into its plant growth-promotion and biocontrol properties. Deletion of the slrR gene, a key regulator of biofilm formation, resulted in significant alterations in biofilm structure and development, as demonstrated by scanning electron microscopy and quantitative biofilm assays. These findings provide valuable insights into the mechanisms of biofilm formation, which are critical for root colonization and plant growth promotion. Additionally, we overexpressed the bac gene cluster responsible for bacilysin biosynthesis by replacing its native promoter with the strong constitutive promoter P43 and integrating an additional copy of the bacG gene. This genetic manipulation led to a 2.7-fold increase in bacB gene expression and significantly enhanced antibacterial activity against Escherichia coli and Lactobacillus diolivorans. The successful implementation of the CRISPR-Cas9 system for genome editing in FZB42 provides a valuable tool for genetic engineering, with the potential to improve its biocontrol efficacy and broaden its applications in agriculture and other biotechnology areas. Our principles and procedures are broadly applicable to other agriculturally significant microorganisms.