Abstract: To investigate the biological control of tobacco brown spot (TBS), culturable bacterial strains were isolated from tobacco leaves and subsequently screened for effective antagonistic strains against Alternaria alternata. The plate face-off test demonstrated that strain WN1 exhibited an inhibition rate of 84.62% on the hyphal growth of A. alternata. Furthermore, the fermentation liquid derived from WN1 significantly inhibited conidial germination of A. alternata. The broad-spectrum antibacterial activity assay revealed that WN1 also possessed a pronounced inhibitory effect on several important plant pathogens, including Valsa mali, Colletotrichum gloeosporioides, Magnaporthe oryzae, Fusarium graminearum, Phytophthora infestans, and P. parasitica. Morphological identification, along with physiological and biochemical analyses, as well as phylogenetic analysis, confirmed that strain WN1 belongs to Bacillus methylotrophicus. Microscopic observation revealed that the WN1-GFP tagged strain was capable of colonizing both the surface and internal tissues of tobacco leaves. The colonization numbers of WN1 were maintained at 10³ CFU/g in the tobacco leaves sprayed with 600 times dilution of WN1 fermentation liquid, monitored at 40 days post-inoculation. The strain WN1 induced and significantly increased the activities of defense-related enzymes in tobacco, such as peroxidase (POD), catalase (CAT), polyphenol oxidase (PPO), and phenylalanine ammonia-lyase (PAL). The field efficacy evaluation demonstrated that the application of WN1 strain fermentation broth at a rate of 6 kg/hm² resulted in a control efficacy of 85.29% against TBS. Considering both the laboratory and field inhibitory effects of WN1 on TBS,WN1 demonstrates significant potential as a promising biocontrol agent for the management of TBS.