Aims The aim of this study was to evaluate the antagonistic activity of halotolerant bacteria against rice brown stripe pathogen Acidovorax oryzae. Methods and Results Fifteen of 136 isolates of halotolerant bacteria exhibited strong in vitro and in vivo antagonistic activity against both strains of A. oryzae. The 15 antagonistic isolates were identified as 'operational group Bacillus amyloliquefaciens' based on physiological and biochemical features, fatty acid profiles as well as sequence analysis of 16S rRNA, gyrA and rpoB genes. Furthermore, this result indicated that the most effective antagonistic isolates K5-3 and PPB6 could produce siderophore in iron-limiting medium, and four kinds of secondary metabolites based on MALDI-TOF analysis. In addition, the culture filtrates of isolates K5-3 and PPB6 caused the damage of cell membrane evidenced by the TEM images, and resulted in 73-80% reduction in cell numbers, 55-65% reduction in biofilm formation, and 42-50% reduction in swimming ability of both strains of A. oryzae. Conclusions These isolates in particular K5-3 and PPB6 of halotolerant bacteria markedly inhibited the growth of A. oryzae. Significance and Impact of the Study To our knowledge, this is the first report on biological control of halotolerant bacteria against bacterial brown stripe of rice.