Abstract: 【Objective】To diagnose an unknown leaf spot disease of tobacco and study its microecological characteristics，which provided scientific basis for the precise prevention and control of this unknown tobacco leaf spot disease. 【Method】Using diseased tobacco leaves with unknown leaf spot disease collected from the tobacco growing region of Shibing in Guizhou as materials，the phyllosphere fungi were isolated，purified and identified by tissue culture method. Based on Koch’s postulates，screening and verification of pathogenic bacteria was conducted by stab inoculation. Highthroughput sequencing technology（ITS and 16S rRNA）was utilized to analyze the phyllosphere microbial communities and diversity.【Result】Four strains of fungi SBR111，SBR121，SBR222 and SBR232 were isolated from the diseased tobacco leaves. Strains SBR111 and SBR121 were identified as Didymella，and strains SBR222 and SBR23 were identified as Hypoxylon by molecular identification. According to Koch’s postulates，strain SBR121 was confirmed as the pathogenic fungus，and morphological and molecular identification classified it as Didymella segeticola. The phyllospheric dominant fungi phyla of diseased and healthy leaves all were Ascomycota，Basidiomycota and Mortierellomycota，and the phyllospheric dominant bacteria phyla of diseased and healthy leaves all were Proteobacteria and Firmicutes. For diseased leaves，the dominant genera of fungi were Phoma，Alternaria and Boeremia，while those of bacteria were Pseudomonas，unidentified_Rhizobiaceae，Stenotrophomonas and Sphingomonas. For healthy leaves，the dominant genera of fungi were Alternaria，Phoma，Boeremia and Hannaella，and those of bacteria were Pseudomonas，Sphingomonas，unidentified_Rhizobiaceae and Methylobacterium. For phyllosphere fungi Alpha diversity index of healthy tobacco leaves， except that the coverage index was equal to that of diseased tobacco leaves，diversity index and abundance index were higher than diseased tobacco leaves. The phyllosphere bacteria diversity index and abundance index of healthy tobacco leaves were lower than that of diseased tobacco leaves. The microbial community functional prediction results indicated that phyllosphere fungi in diseased leaves were mainly distributed in plant_pathogens-wood_saprotroph fungi group， while the phyllosphere fungi in healthy leaves were mainly distributed animal_pathogen-endophyte-plant_pathogenwood_saprotroph and endophyte-plant_pathogen groups；the phyllosphere bacteria of diseased and healthy tobacco leaves were mainly distributed in 3 pathways：metabolism，genetic information processing and environmental information processing.【Conclusion】Didymella segeticola is the pathogen of tobacco leaf spot in Shibing tobacco growing region of Guizhou. There are differences in the community structure and diversity of phyllosphere fungi and bacteria between healthy and diseased tobacco leaves. The diversity and richness indexes of phyllosphere fungi in diseased tobacco leaves are lower than those in healthy tobacco leaves，and the diversity index and richness index of phyllosphere bacteria are higher than those in healthy tobacco leaves.