Abstract: 12% difenoconazole + fluxapyroxad SC (commercial name: Jiangong) was first released by BASF in China in 2016. It has been registered to control many diseases, including pear scab, apple Alternaria leaf spot, tomato early blight, cucumber powdery mildew, etc. This study evaluated the bioactivity of Jiangong against Alternaria alternata and explored variations of phyllosphere microorganisms in both asymptomatic and tobacco brown spot leaves at different persistence periods (0, 5, 10, and 15 days post-fungicide application) using high-throughput sequencing technology. The results indicated that Jiangong effectively inhibited mycelial growth (average EC₅₀ value of 0.51 μg/mL), conidia germination (3.47 μg/mL), and the carbon metabolism of A. alternata. Both asymptomatic and brown spot leaves presented complex microbial communities. Higher fungal diversity was noted in asymptomatic leaves, while higher bacterial diversity was found in symptomatic leaves. After application, the diversity and abundance of microbial community structures in both types of leaves changed over time. Fungal microbiome communities showed greater sensitivity than bacterial groups, with the microbiome communities of asymptomatic leaves being more affected than those of symptomatic leaves. Fungal community diversity decreased for both symptomatic and asymptomatic leaves after 5 days of application, while the diversity of fungal community in symptomatic leaves showed an upward trend after 10 days of application. Meanwhile, bacterial community diversity increased in both symptomatic and asymptomatic leaves after 5 days of application but then declined in asymptomatic leaves after 15 days. The abundance of the dominant function group of phyllosphere bacteria (metabolism, genetic information processing, environmental information processing) was not affected by the application of the mixture. However, the abundance of the dominant function group of phyllosphere fungi (pathogen-endophyte-wood saprotroph, endophyte-plant pathogen, plant pathogen-undefined saprotroph) was significantly affected by the application of the mixture, and high variation was found in symptomatic leaves than that of asymptomatic leaves. The application of Jiangong-induced alterations in the community structure of the tobacco phyllosphere microbiome provides a basis for future tobacco brown spot control strategies based on phyllospheric microecology.