Natural rubber is an important national strategic and industrial raw material. Leaf anthracnose of rubber tree caused by the Colletotrichum species is one of the important factors restricting the yield of natural rubber. In this study, we isolated and identified strain Bacillus velezensis SF334 that exhibited significant antagonistic activity against both of C. australisinense and C. siamense, the dominant species of Colletotrichum causing leaf anthracnose of rubber trees in Hainan province of China, from a pool of 223 bacterial strains. The cell supernatants of SF334 had a significant prevention effect for leaf anthracnose of rubber tree with an efficacy of 79.67% against C. siamense and 71.8% against C. australisinense, respectively. We demonstrated that SF334 can lead to the lysis of C. australisinense and C. siamense mycelium by causing mycelial expansion, resulting in mycelial rupture and subsequent death. B. velezensis SF334 also harbors some traits of plant growth-promoting rhizobacterium such as secreting siderophore, protease, cellulose, pectinase and auxin of indole-3-acetic acid (IAA), as well as has broad-spectrum antifungal activity against some important plant pathogenic fungi. The genome combined with comparative genomic analyses indicated that SF334 possesses most genes of the central metabolic and secondary metabolites of B. velezensis strains. To our knowledge, this is the first time to report a Bacillus velezensis strain as a promising biocontrol agent against leaf anthracnose of rubber tree caused by C. siamense and C. australisinense. The results suggest that B. velezensis could be a potential candidate agent for leaf anthracnose of rubber tree.