Abstract: Tobacco target spot is a fungal disease on tobacco. In this paper, the mycelial growth rate method was used for the potential evaluation of dimetachlone to Rhizoctonia solani, and the effect of dimetachlone on the microbes of tobacco leaves after different periods of application was studied with Illumina high-throughput sequencing technology as well. Results showed that dimetachlone had strong inhibitory activity against the mycelial growth of R. solani with an EC₅₀ value of 1.20 μg/mL and a complete inhibition of mycelial growth at 6.47 μg/mL. Within 0-18 days of treatment with 4200 g/hm² dimetachlone 40% WP, there were significant differences in microbial community structure between healthy and diseased tobacco leaves. The phyllosphere fungi in both healthy and diseased tobacco were all distributed in Thanatephorus, Alternaria, and Fusarium, the phyllosphere bacteria in tobacco leaves were Pseudomonas, Staphylococcus, and Sphingomonas. Three days after application, the relative abundance of Thanatephorus in healthy and diseased tobacco leaves decreased 12.41% and 51.62%, the relative abundance of Alternaria increased 0.54% and 0.42%, the relative abundance of Pseudomonas decreased 13.48% and 19.17%, respectively. Nine days after application, the relative abundance of Thanatephorus in healthy and diseased tobacco leaves decreased 1.38% and 47.42%, the relative abundance of Alternaria decreased 0.36% and 0.18%, the relative abundance of Pseudomonas decreased 2.73% and 2.73%, respectively. Eighteen days after application, the relative abundance of Thanatephorus in healthy and diseased tobacco leaves decreased 26.74% and 39.03%, the relative abundance of Alternaria increased 26.02% and 2.70%, the relative abundance of Pseudomonas increased 6.56% and 16.02%, respectively. The application of dimetachlone 40% WP in the field significantly suppressed the relative abundance of Thanatephorus genus in healthy and diseased tissues within 3 d, but the effect on disease tissues was greater than that on healthy leaves; it also caused changes in the structure of interleaf bacterial flora, but the effect on diseased leaves was less than that on healthy tobacco leaves. The results of the study revealed the differences in the phyllosphere microorganism communities between healthy and diseased interleaf tissues after the application of dimetachlone from a microscopic perspective, providing a scientific basis for the application of dimetachlone.