Abstract: To evaluate the effects of S-metolachlor on soil microbial communities and metabolic functions in flue-cured tobacco fields, this study investigated temporal changes in bacterial and fungal community structures, diversity, and carbon source metabolism within 60 days after application of S-metolachlor emulsifiable concentrate (EC, 1012 mL/hm²) using Illumina amplicon sequencing and Biolog-ECO techniques. The results indicated that soil fungal richness remained unaffected during the initial 40 days after application, while fungal diversity showed no significant variation within the first 10 days. Notably, both bacterial and fungal diversity and richness increased significantly after 60 days. At the phylum level, Actinobacteriota and Proteobacteria dominated bacterial communities, whereas Ascomycota prevailed among fungi. Genus-level analysis revealed significant increases in Gemmatimonas and Flavobacterium abundance, while Sphingomonas exhibited fluctuating patterns. Fungal communities demonstrated significant enrichment in Mortierella and Botryotrichum, contrasting with a continuous decline in Actinomucor. Carbon metabolism profiling revealed enhanced microbial utilization of 31 carbon sources after treatment, with particularly pronounced metabolic activation in nine substrates including putrescine and α-ketobutyric acid. These results demonstrate that S-metolachlor exerts no significant toxicity or inhibitory effects on soil microbiota in flue-cured tobacco fields, instead exhibiting stimulatory effects on microbial growth. This study elucidates the regulatory mechanisms of S-metolachlor on soil microecology and provides a scientific basis for its rational application in agricultural practices.