Abstract: To effectively control and prevent bacterial wilt of cigar tobacco in Hainan, strains with antagonistic activity against Ralstonia solanacearum were isolated from the rhizosphere soil of cigar tobacco using tissue homogenate method and plate confrontation method, followed by molecular and phenotypic identification. Furthermore, the medium components and fermentation conditions of these antagonistic strains were optimized, and their biocontrol efficacy and growth-promoting effects were verified via pot experiments. The results showed that: (1) Three strains (HNC-3, HNC-6 and HNC-17) with significant antagonistic activity against Ralstonia solanacearum of cigar tobacco were screened out, which were identified as Bacillus velezensis, B. safensis and B. subtilis, respectively. The diameters of their inhibition zones were (30.12±1.12) mm, (25.81±1.23) mm and (31.56±0.64) mm, respectively. (2) The optimal fermentation conditions for the three highly effective antagonistic strains were clarified. Taking HNC-3 as an example, its optimal fermentation conditions were determined as follows: Glucose as the carbon source, bacteriological peptone as the nitrogen source, sodium dihydrogen phosphate as the inorganic salt, initial pH 7.0, a shaking speed of 190 r/min, an inoculum size of 3% and a liquid volume of 15%. After the optimization of fermentation medium and conditions, the bacterial concentrations of the three strains was significantly increased, and the diameter of inhibition zone was also significantly enlarged. (3) Pot experiment results indicated that the disease control efficacies of HNC-3 and HNC-17 against cigar tobacco bacterial wilt reached 70.35% and 74.59%, respectively, which were significantly superior to that of thiodiazole copper. (4) All the three biocontrol strains could significantly promote the growth of tobacco seedlings; in particular, the plant height, root length and fresh mass of above-ground parts in the HNC-6 treatment group were increased by 13.4%, 27.6% and 56.6% compared with those in the control group, respectively. The biocontrol strains screened in this study lay a solid foundation for the development of biocontrol microbial agents and the implementation of green prevention and control strategies against bacterial wilt of Hainan cigar tobacco.