Abstract: To clarify the biodiversity, biocontrol and growth-promoting effects of endophytic bacteria in tobacco (Nicotiana tabacum), 17 strains of endophytic bacteria were isolated from tobacco leaves using traditional isolation and culture methods. Morphological observation, Gram staining, 16S rDNA sequencing and phylogenetic tree construction were conducted to determine their biocontrol effects, and pot experiments were conducted to investigate the growth-promoting effects of the microbial agents. The results of Gram staining showed that five strains were Gram-positive and 12 strains were Gram-negative. Morphological observations showed that 11 strains were rod-shaped and six strains were spherical. 16S rDNA sequencing and BLAST comparative analyses indicated that strains YC01, YC11, YC12 and YC13 were Bacillus; strains YC02, YC03, YC04, YC06, YC07, YC15, YC16 and YC17 were Pseudomonas; strain YC05 was Acinetobacter; strain YC08 was Rosenbergiella; strain YC09 was Klebsiella; strain YC10 was Stenotrophomonas, and strain YC14 was Microbacterium. The results of the phylogenetic tree analysis showed that the 17 strains could be grouped into 10 major branches. The biocontrol confrontation test showed that the 17 strains exhibited different inhibitory effects on six common pathogenic fungi of tobacco, and had a broad antibacterial spectrum. All 17 strains showed the strongest inhibitory effect on Phytophthora nicotianae, achieving an average antibacterial rate of 60.64%. Evaluation of the growth-promoting potential showed that 10 endophytic strains were capable of producing protease and indoleacetic acid, four strains were found to have nitrogen-fixing ability, 12 strains were capable of producing siderophores, and 14 strains demonstrated the ability to solubilize inorganic phosphorus. Strains of Bacillus, Pseudomonas, Rosenbergia and Klebsiella that exhibited high growth-promoting potential were selected for subsequent growth-promoting pot experiments. The results showed that four of the microbial agents appeared to promote tobacco growth. The application of the microbial agents resulted in significant increases in chlorophyll and nitrogen contents, fresh biomass, and dry biomass of both aerial parts and roots of tobacco plants, thereby significantly enhancing the growth and root development of tobacco seedlings, among which the effect of microbial agents of Bacillus was the most pronounced.