Bacterial wilt caused by Ralstonia solanacearum (Rs) is one of the most serious tobacco diseases worldwide. An organic fertilizer was secondly solid fermented by two antagonists, making it a bio-organic fertilizer (BOF). The BOF was made up of BOF25 fermented by strain L-25 and BOF9 fermented by strain L-9 respectively in a 1:1 (w:w) proportion. Field experiments were conducted in Anhui Province for two years to investigate the biocontrol efficacy of BOF. Results showed that the control efficacies of the BOF treatment were up to 75.2% for the first year and 95.4% for the second year. The results of scanning electron microscope (SEM) observations showed that there was viscous material deforming and blocking vessels in the vascular bundles of wilted tobacco, while the vascular bundles of healthy tobacco and those treated with BOF grew well with a normal shape. The populations of cultivated bacteria, actinomycetes and antagonists were significantly higher than in the control, while the number of fungi significantly decreased. BOF depressed the colonization of Rs on tobacco roots when Rs was labeled by green fluorescent protein. The functional diversity of the microbial community, as determined by the Shannon Index, Simpson Index, and Mclntosh Index of the soil microbial community, were significantly higher in the BOF treatment than in the control. The DGGE patterns of bacteria and fungi from the BOF soil and control treatments belonged to two corresponding clusters, suggesting that bacterial species increased and fungal species decreased with the application of BOF. These results suggest that the tobacco-specific BOF application can effectively improve the micro-ecology in the rhizosphere, and is thus a potentially promising treatment for the control of tobacco bacterial wilt disease.