Abstract: In order to effectively control tobacco brown spot disease, this study aimed to screen an optimal ratio of biological and chemical agents for synergistic inhibition of tobacco brown spot disease pathogen (Alternaria alternata). Using mycelial growth rate method, the bioactivity of 40% dimetachlone and Bacillus amyloliquefaciens Cas02 against A. alternata was determined. The compatibility between the two agents was assessed by plotting growth curves, and the joint toxicity and potted control efficacy of different combination ratios of the two agents were evaluated. The results indicated that both 40% dimetachlone and B. amyloliquefaciens Cas02 had high toxicity against A. alternata, with EC₅₀ values of 4.86 μg/mL and 4.6×10⁷ cfu/mL, respectively. Compatibility test showed that Cas02 was not sensitive to sclerotium resistance, allowing for their combined use. The best synergistic effect on tobacco brown spot disease was observed with the combination ratio (V/V) of 40% dimetachlone and B. amyloliquefaciens was 7∶3, resulting in an enhancement ratio of 1.32 and an actual inhibition rate of 67.61%. Potted experiments demonstrated that the 7∶3 volume ratio combination of 40% dimetachlone and B. amyloliquefaciens had the best control efficacy against tobacco brown spot disease, achieving 74.12%, which was higher than that of the single-agent treatments. Therefore, it is recommended to mix 4.86 μg/mL of 40% dimetachlone and 4.6 × 10⁷ cfu/mL of B. amyloliquefaciens Cas02 at a ratio of 7∶3 for the control of tobacco brown spot disease in field.