Three out of 203 bacterial isolates showed high antagonistic activity by in vitro screening against three strains of Xanthomonas spp. causing leaf spots from Rosa spp.. Antagonistic bacteria were collected from the rhizosphere substrate of potted rose in Sa Dec Flower Village of Dong Thap province, Viet Nam. While three pathogen strains of Xanthomonas spp. were stored at the Biochemistry Laboratory of Biotechnology Research and Development Institute of Can Tho university. Three effective isolates were identified by Matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) and analysis of 16S rRNA gene sequence. Using MALDI-TOF, these antagonistic bacteria belong to Bacillus genera. The amplification of 16S rDNA gene was performed using 27F and 1492R primers. The nucleotide sequences of this gene were aligned using the GenBank database and BLAST-N program from the NCBI site. The isolates identity of BR16, BR37, and BR88 shared the highest similarity values in turn with Bacillus velezensis MN160320 (99.11%), Bacillus subtilis MN493770 (99.11%), Bacillus amyloliquefaciens KX871898 (99.41%). These Bacillus isolates were designed Bacillus velezensis MW677565 (from BR16), B. subtilis MW828613 (from BR37), B. amyloliquefaciens MW828656 (from BR88). These isolates have shown the ability to fight phytopathogenic bacteria of rose plants in Dong Thap of Viet Nam.