As one of the most common and destructive diseases, tobacco wildfire disease, caused by Pseudomonas syringae pv. Tabaci, results in significant losses every year in Chinese tobacco growing areas. In order to develop biocontrol agents against tobacco wildfire, 14 strains that have antagonism against tobacco wildfire were screened out of 278 isolates separated from tobacco leaves and identified as Bacillus subtilis based on 16S rDNA sequence. The inhibition zone of the most antagonistic strains against tobacco wildfire were 9.3 mm. After spraying of the antagonistic bacteria, the activity of phenylalanine ammonialyase (PAL), peroxide enzyme (POD) and the polyphenoloxidase (PPO) activeness in the tobacco leaf were increased significantly. The results of the efficiency test confirmed that when the plants were covered with antagonistic bacteria before inoculation of pathogen bacteria, the control efficacy to tobacco wildfire reached 97.54%, which is significantly higher than that of agro-streptomycin. And the control efficacy was 51.40% when the plants were sprayed with antagonistic bacteria after inoculation of the pathogen bacteria.