Abstract: Polyoxin is a fungicide used to prevent and control leaf spot disease in tobacco. To investigate the changes in phyllosphere microecology after polyoxin application, Illumina high-throughput sequencing was used to analyze the microflora structure and diversity of phyllosphere microorganisms at different times after polyoxin application, and Biolog-ECO was used to analyze the metabolic function of phyllosphere microorganisms. The results showed that the phyllosphere fungi in both healthy and diseased tobacco leaves were mainly distributed in Ascomycota (64.20%, 94.05%) and Basidiomycota (6.77%, 2.40%). The bacteria were mainly distributed in Proteobacteria (7.16%, 38.81%) and Firmicutes (16%, 0.78%). The diversity index of phyllosphere fungi and bacteria in healthy tobacco leaves was significantly higher than that in diseased tobacco leaves. The phyllosphere microbial metabolic activity of healthy tobacco leaves was stronger than that of diseased tobacco leaves. After the application of polyoxin, the relative abundances of Alternaria, Boeremia, Pantoea, and Pseudomonas in diseased tobacco leaves decreased first and then increased. The relative abundances of Cladosporium, Epicoccum, Didymella, Symmetrospora, Stagonosporopsis, and Sphingomonas in diseased tobacco leaves increased first and then decreased. The relative abundances of Alternaria, Epicoccum, Symmetrospora, Stagonosporopsis, and Sphingomonas in healthy tobacco leaves increased first and then decreased, while the relative abundances of Cladosporium, Boeremia, Pantoea, and Pseudomonas in healthy tobacco leaves generally decreased. The diversity of phyllosphere fungi and bacteria in diseased tobacco leaves was on the rise, while fungi diversity in healthy tobacco leaves was on the rise and bacteria diversity was on the decline. In terms of metabolic function, after the application of polyoxin, the metabolism of 31 kinds of carbon sources of diseased and healthy phyllosphere microorganisms was inhibited to varying degrees, and the metabolic activity gradually recovered with the time of application. The results revealed the changes in phyllosphere microflora structure and diversity after the application of polyoxin and provided a reference for the study of the microecological mechanism of plant disease prevention and control by fungicides. The effect of polyoxin on the microflora structure and microbial metabolism of healthy tobacco leaves is higher than that of diseased tobacco leaves, suggesting that polyoxin has the best efficacy as a protective agent in production.