Abstract: To determine the sensitivity level of tobacco brown spot to the new fungicide pydiflumetofen and the safety of this agent in tobacco leaves and soil, the sensitivity of Alternaria. alternata to pydiflumetofen was determined by mycelium growth rate method and spore germination method. Field control tests were carried out in Yunnan, Sichuan, Guizhou, Fujian and Chongqing, and we analyzed the residual degradation of pydiflumetofen in tobacco leaves. Meanwhile, high-throughput sequencing was used to analyze its effects on community structure and co-occurring network of soil bacteria and fungi. The results showed that pydiflumetofen had strong inhibitory activity against A. alternata at different developmental stages, and the mycelium growth stage was the most sensitive. The EC₅₀ range of pydiflumetofen for 169 A. alternata infected strains was 0.0064~0.1832 mg/L. Field experiments showed that the efficacy of pydiflumetofen suspension applied twice on tobacco brown spot ranged 69.42% to 88.09%, and the tobacco yield was increased. In addition, the degradation half-life period of pydiflumetofen in fresh and dry tobacco leaves ranged 3.2~8.8 d. The beneficial microbes such as Streptomyces, Pseudomonas, Trichoderma and Paecilomyces were significantly enriched after the use of pydiflumetofen, and the complexity of bacterial and fungal cogenetic network increased. Therefore, pydiflumetofen can be used for the prevention and control of tobacco brown spot.