Abstract: To explore multifunctional beneficial microbial strains that promote tobacco growth, rhizosphere soil samples from healthy crops in the central Henan Province were collected. Bacterial strains with growth-promoting activities, such as IAA production and antibacterial capabilities, were isolated and screened. Subsequently, these strains were identified through morphological, physiological, biochemical, and 16S rDNA sequence analyses. The growth-promoting effects of the strains were further verified through seed germination experiments in Petri dishes and inoculation in floating seedling substrates. The results indicated that the isolation and purification yielded rhizosphere soil bacterial JHS0317 and XL0404, which could produce IAA up to 26.81 and 18.94 mg/L, respectively, and ACC deaminase up to 470.34 and 484.48 U/L, respectively. Both strains exhibited functions in phosphate solubilization, potassium release, nitrogen fixation, and siderophore production. Additionally, they showed inhibitory effects on multiple plant pathogens, with inhibition rates ranging from 31.94% to 55.88%. Upon identification, strain JHS0317 was determined to be Pantoea agglomerans, while strain XL0404 was identified as Enterobacter ludwigii. Both strains JHS0317 and XL0404 enhanced the germination rate of tobacco seeds and significantly promoted root growth. When the concentration of strains JHS0317 and XL0404 in the seedling substrate was 1×10⁵ cfu/g, the root activity of tobacco seedlings was at its highest. When the concentrations of the two strains were 1×10⁵ and 1×10⁷ cfu/g, respectively, the fresh weight of the whole plant, above-ground fresh weight, root fresh weight, maximum leaf length, and maximum leaf width of tobacco seedlings were all significantly higher than those of the control treatment. Compared to the control, these values increased by 153.73%, 171.93%, 40.00%, 54.06%, 56.17%, and 66.42%, 65.79%, 70.00%, 33.19%, 26.17%, respectively. In summary, the two plant growth-promoting rhizobacteria, JHS0317 and XL0404, exhibited significant growth promoting effects on tobacco plants, indicating their potential importance in enhancing tobacco leaf yield and quality. Additionally, they demonstrated promising biocontrol potential.