Abstract: Soil microbial communities play a critical role in maintaining soil health and suppressing soil-borne diseases. This study analyzed the bulk soils of healthy and Fusarium root rot-infected tobacco plants from Longshan and Guandiping in Hunan Province, combining soil physicochemical properties and high-throughput sequencing to investigate microbial community composition, structural differences, functional predictions, and key differential taxa. The results showed that: (1) Within the suitable pH range for tobacco growth, diseased soils exhibited significantly lower pH but higher ammonium (NH₄⁺-N) and nitrate (NO₃⁻-N) content compared to healthy soils. (2) Fungal diversity significantly decreased in diseased soils, while fungal richness increased; bacterial diversity and richness showed no significant differences. (3) Pathogenic and saprophytic fungal functional groups were enriched in diseased soils. (4) LEfSe analysis further identified nine genera (e.g., Botryotrichum) as biomarkers of healthy soils, showing negative correlations with NH₄⁺-N and NO₃⁻-N but positive correlations with pH. Conversely, seven genera (e.g., Trichocladium) were identified as biomarkers of diseased soils, exhibiting positive correlations with NH₄⁺-N and NO₃⁻-N but negative correlations with pH. In conclusion, within the suitable pH range, the co-occurrence of relative acidification (pH reduction), nitrogen enrichment (particularly NH₄⁺-N and NO₃⁻-N), and microbial community imbalance (e.g., pathogen/saprophyte enrichment, biomarker shifts) synergistically drove the occurrence of Fusarium root rot in tobacco.