Abstract: The objective of this study was to identify the pathogen and screen the biological control agents of tobacco leaf mold, which causes damage losses during the yellowing stage of leaf curing each year in several counties of Dali Bai Autonomous Prefecture of Yunnan Province, China. Infected tissues were used for isolating the causal pathogen, which were classified by combining results from the morphology and multiple gene sequences analysis. Antagonistic activities of bacteria against the pathogen were evaluated in vitro and in vivo. Fungi obtained from infected tissues were grouped into the genus Rhizopus based on morphological characteristics. Furthermore, internal transcribed spacer region (ITS) sequences of isolates had the highest similarity with R. arrhizus subsp. arrhizus CBS 112.07^T. Similar results were obtained from multiple sequence alignment and phylogenetic analyses using the partial sequences of ITS, actin (ACT), translation elongation factor 1-α (TEF), and the largest subunit of RNA polymerase II (RPB1). Above all, the pathogen causing tobacco leaf mold in Dali is R. arrhizus. Besides, five antagonistic bacteria had robust inhibition activities against hyphal growth of R. arrhizus in vitro. Among these bacteria, the Bacillus strains Z002 and 05-1205 were selected for biological control efficiency tests in the barn and showed strong evidence to support them as potential biological agents with relative control efficiency >70%.