Finding synthetic pesticide alternatives for health and a healthy environment has become a crucial issue for scientific research. A number of studies have reported efficacy of Bacillus species on promoting plant development, as well as protecting plants against pathogen invasion, especially pathogenic fungi and bacteria. However, little was known about Bacillus species in controlling viral diseases. In this study, Bacillus atrophaeus strain HAB-5, isolated from cotton field, Xinjiang, China efficiently promoted the growth of tobacco plants. According to the results, the treatment with the strain HAB-5 increased the expression of NtEXP1 and NtEXP2. Then the Tobacco mosaic virus (TMV)/ Nicotiana tobacco system was employed to evaluate virus resistance induced by strain HAB-5. Tobacco leaves were treated with antimicrobial metabolites of strain HAB-5 (1 mg/mL), and 12 h later the treated leaves were challenged with TMV via rub-inoculation. The results showed that disease symptoms were obviously compromised by tobacco leaves treated with strain HAB-5, and the viral accumulation level was reduced extensively. Moreover, it was found that the signaling regulatory gene (NPR1), defense genes (PR-1a, PR-1b, Chia5), and hypertensive response related genes (Hsr203J, Hin1) were up-regulated in plants treated with the metabolites. Altogether, these accumulated results strongly support strain HAB-5 to be a biological controlling agent against TMV.