Bacterial wilt caused by Ralstonia solanacearum is one of the most destructive diseases which negatively affects the tomato production worldwide. Considering the importance of the disease, using biological control agents could be effective approaches in reducing the damage of the pathogen. Plant specimens suspected to be infected by tomato bacterial wilt disease were collected from different regions of East Azerbaijan province in Iran. Phenotypic identification of the strains was performed using recommended biochemical and physiological tests. In genotypic assessment, using PCR, a 282 and 148 bp fragments with the PS96I/ PS96H primers were amplified. Based on phenotypic and genotypic properties, the strains that cause wilt of tomato were identified as R. solanacearum biovar 2. For biological control of this disease, 15 bacterial strains were collected from rhizosphere of healthy tomato plants using soil serial dilution method. Antagonistic effect of the strains was evaluated based on their growth inhibitory on pathogen. According to the results, three bacterial strains showed antagonistic activity against R. solanacearum. Based on phenotypic and genotypic characteristics, the antagonist strains Ka1, Ka2 and Ka3 were identified as Bacillus subtilis and Pseudomonas fluorescens, respectively.