Abstract: To screen and identify efficient biocontrol bacterial strains against tobacco root rot caused by Aphanomyces iridis, the antagonistic effects of Bacillus velezensis Ba-0321, Paenibacillus polymyxa LB-9 and Trichoderma asperellum Tr-0111 were determined by plate confrontation cultivation. The inhibitory effects of bacterial solutions, fermentation broths and volatile metabolites of the three biocontrol strains on mycelial growth of A. iridis were determined using mycelial growth rate methods. The control effects of the fermentation broths of these strains on tobacco root rot caused by A. iridis were analyzed by inoculation experiments using floating seedlings. The results showed that all three biocontrol strains had antagonistic effects on A. iridis. When the solution concentrations of B. velezensis Ba-0321, P. polymyxa LB-9 and T. asperellum Tr-0111 were 1 × 10⁷, 1 × 10⁸ and 1 × 10⁶ cfu/mL, the inhibition rates on mycelial growth of A. iridis were 86.70%, 85.32% and 92.63%, respectively. Among the tested strains, T. asperellum Tr-0111 had the strongest inhibitory effect against A. iridis with an EC₅₀ of 3.59 × 10² cfu/mL. The EC₅₀ values of B. velezensis Ba-0321 and P. polymyxa LB-9 were 1.74 × 10⁴ and 1.41 × 10⁵ cfu/mL, respectively. The fermentation broths and volatile metabolites of the three biocontrol strains also had inhibitory effects on the mycelial growth of A. iridis, with those of B. velezensis Ba-0321 showing the highest inhibition rates of 42.73% and 16.31%, respectively. In the floating seedling inoculation experiment, the relative control effect of the fermentation broth of T. asperellum Tr-0111 on tobacco root rot caused by A. iridis was 66.53%, significantly (P < 0.5) higher than that of B. velezensis Ba-0321 (51.69%) and P. polymyxa LB-9 (40.95%).