Abstract: In order to screen bacterial strains that can efficiently antagonize major molds in the storage of cigar tobacco stacks, and to enrich the microbial resource bank for biological control of cigar tobacco leaf mildew, strains were isolated and purified from non-mildewed tobacco leaves in the workshop of Great Wall Cigar Factory, China Tobacco Sichuan Industrial Co., Ltd. Five species of mold-causing fungi, Aspergillus cristatus, Syncephalastrum monosporum, Aspergillus chevalieri, Scopulariopsis brevicaulis and Aspergillus montevidensis, which were previously isolated in the laboratory from mildewed cigar tobacco leaves during the stack storage period, were used as the target strains. Strains with broad-spectrum antagonistic activity against all five pathogenic fungi were screened using the agar diffusion method, and the antagonistic strains were identified based on morphological, physiological and biochemical, and molecular biological characteristics. The results were as follows (1) 3 strains TB24016, TB24034, TB24048 had excellent antagonistic effects on five molds. Strains TB24016 and TB24048 were identified as Bacillus velezensis, and TB24034 as Bacillus halotolerans; (2) All 3 antagonistic strains (TB24016, TB24034 and TB24048) exhibited strong antagonistic effects against A. chevalieri, with inhibition zone diameters of 4.49, 3.46 and 2.27 cm, respectively; (3) Strains TB24016 and TB24034 produced protease and siderophore, whereas strain TB24048 produced only siderophore. The extracellular enzyme production capacity ranked as TB24016 > TB24034 > TB24048; (4) The inhibitory effects of fermentation broths from different antagonistic strains varied among the molds isolated from tobacco leaves. When the concentration of fermentation broth was 10⁸ CFU/mL, all three antagonistic strains demonstrated inhibition rates exceeding 80% against the five tested molds, with overall antifungal performance ranked as: TB24016 ≈ TB24048 > TB24034. In conclusion, based on the extracellular enzyme-producing ability and mold-inhibiting effect, Bacillus velezensis TB24016 exhibited superior antagonistic effects against dominant mold-causing fungi in cigar tobacco during stack storage, demonstrating strong potential as a biocontrol agent for mold control in cigar tobacco leaves.