Abstract: This study aimed to investigate the culturable microbial diversity in the tobacco rhizosphere microenvironment under different health conditions and to screen for beneficial strains for the biological control of bacterial wilt. Using cultivation-based and molecular techniques, we compared rhizosphere microbiota associated with different ecological niches (rhizosphere soil, root surface soil and root tissue) of healthy and bacterial wilt-infected tobacco plants. The isolated strains from these distinct niches were evaluated for their antagonistic activity against the bacterial wilt pathogen and their plant growth-promoting (PGP) functions, with the most promising candidates subsequently validated in seed germination assays and pot experiments for their efficacy in disease suppression and growth promotion. The results showed that a total of 370 bacterial strains were isolated from the rhizosphere of both healthy and bacterial wilt-infected tobacco plants, including 215 strains originating from healthy samples and 155 from diseased samples. The diversity of culturable bacteria in rhizosphere soil and on root surface soil was greater than that in root tissues. Molecular identification indicated that Pseudomonas and Bacillus were significantly enriched in healthy plants, whereas the relative abundance of Variovorax and Paenibacillus was markedly higher in diseased plants. Among the isolates, 89 representative strains were tested for antagonistic activity against Ralstonia solanacearum, the causal agent of bacterial wilt, of which 24 strains exhibited clear antagonistic activity. Subsequently, seed germination assays demonstrated that strains Bacillus velezensis Cas367, Paenarthrobacter nicotinovorans Cas623, Pseudarthrobacter enclensis Cas111, and Pseudomonas rhodesiae Cas489 significantly enhanced tobacco seed germination. Evaluation of plant growth-promoting traits showed that several strains possessed beneficial capabilities, including phosphate solubilization (six strains), potassium solubilization (three strains), and the production of proteases (sixteen strains), amylases (five strains), and siderophores (eleven strains). Based on a comprehensive evaluation of these antagonistic and PGP traits, strains Cas367 and Cas489 were selected for pot experiments. The results showed that both strains significantly promoted plant growth and decreased the disease incidence. Moreover, their co-inoculation provided superior suppression of tobacco bacterial wilt, resulting in a 60.0% lower disease incidence and significantly reducing the disease index compared to the control. This study provides a robust theoretical foundation for the future development of targeted microbial inoculants and synthetic communities against tobacco bacterial wilt.