Abstract: In order to explore the regulatory mechanism of Ralstonia solanacearum stress during the seedling stage on the resistance of tobacco plants, flue-cured tobacco cultivar Honghuadajinyuan was used as the test material, and five concentration gradients 0 (CK), 10² (T1), 10⁴ (T2), 10⁶ (T3), and 10⁸ (T4) CFU/g (dry substrate) were established. The incidence of tobacco seedlings, agronomic traits, enzyme activities of the phenylpropanoid biosynthesis pathway, root activity, and rhizosphere soil microbial community structure were determined. The results showed that after the first inoculation, the incidence rates of T3 and T4 reached 9.20% and 91.59%, respectively. Compared with CK, the fresh mass and dry mass of tobacco seedlings in the T2 treatment were 12.00% and 18.92% higher, respectively. As the inoculation concentration increased, the SPAD value first increased and then decreased; the activities of key enzymes in the phenylpropanoid biosynthesis pathway, such as phenylalanine ammonia-lyase (PAL) and cinnamic acid-4-hydroxylase (C4H), continuously decreased, whereas the activity of 4-coumarate-CoA ligase (4CL) first increased and then decreased. Root activity gradually decreased, with the polyphenol oxidase (PPO) activity inhibited. After the second inoculation, the incidence rates of T1 and T2 were 30% and 20%, respectively, which were significantly lower than 40% of CK, and T2 exhibited the optimal growth and development. The fresh mass of the aboveground parts of non-infected tobacco seedlings in T2 was 11.34% higher than that of CK. The number of soil OTUs in T1 and T2 was significantly higher than that in CK; the abundance of Proteobacteria and Actinobacteria increased, whereas that of Gemmatimonadetes decreased. The Shannon and Chao1 diversity indices of CK were the lowest, and the microbial community structures of the treatment groups were significantly distinct from that of CK. In conclusion, a moderate concentration (10⁴ CFU/g) of R. solanacearum induces systemic acquired resistance (SAR) by enhancing the accumulation of photosynthetic products, optimizing rhizosphere microbial diversity, and regulating carbon metabolism. In contrast, high concentrations (≥10⁶ CFU/g) cause diseases by inhibiting defense enzyme activities and root functions. This study reveals the "concentration threshold effect" of R. solanacearum interference at the seedling stage, providing key parameters for the development of stress-resistant cultivation techniques based on the precise regulation of pathogen concentrations.