Crown gall disease (CGD), which is caused by the bacterium Agrobacterium, is a common plant disease that often results in significant economic losses. Biocontrol offers an environmentally friendly and sustainable method to control CGD. In this study, we investigated the biocontrol effect and mechanism of CGD in Japanese cherry trees by B. velezensis FZB42, a prototype strain of the plant growth promoting rhizobacteria (PGPR). We found that the B. velezensis FZB42 mutants unable to produce one or a combination of four antimicrobial compounds (surfactin, macrolactin, bacilysin, and difficidin) significantly reduced their inhibitory activities against A. tumefaciens XYT-58 and their ability of controlling the occurrence of CGD. Using the purified compounds, we further demonstrated that bacilysin and surfactin can inhibit the growth of XYT-58 in vitro. Inoculation with B. velezensis FZB42 significantly increased the expression of genes related to the jasmonic acid (JA), ethylene (ET), and salicylic acid (SA) pathways in cherry seedlings when they were infected with XYT-58. To our knowledge, this is the first report that bacilysin and difficidin can also suppress the growth of Agrobacterium. Furthermore, our results indicate that multiple antimicrobial compounds and induced systemic resistance (ISR) are involved in the biocontrol of CGD by B. velezensis. The mechanism elucidated here provides guidance for the development and application of this type of PGPR strain in the biocontrol of CGD.