This study reveals the interaction between anthropogenic pressures and stratified microbial ecology in Fuxian Lake (China’s largest deep freshwater reservoir with Class I water quality). By integrating stratification indicators (TN, TP, COD, DO) from surface water with metagenomic sequencing data from surface (0–5 m), mid-depth (20–50 m), and deep-water (50–120 m) zones, we demonstrate how nitrogen loads from tourism activities in the northern shoreline zone trigger genomic adaptive changes in benthic microbial communities. In the coastal zone, dissolved nitrogen (accounting for over 53% of total dissolved nitrogen) drove genomic expansion alongside the spread of tetracycline resistance genes. Conversely, the central deep-water basin exhibited archaeal dominance due to total phosphorus enrichment (0.039 mg/L), showing significantly enhanced genomic integrity and surging alkaline phosphatase expression. This accelerated sediment phosphorus cycling rates to 2.7 times those in surface waters. Microbial communities exhibit depth-stratified functional specialization: (1) Tourism-impaired surface layers (0-5 m) display depressed diversity; (2) Metalimnetic zones (20-50 m) demonstrate ’edge-rich, center-cohesive’ structural continuity (elevated N50/N90 contig metrics centrally); (3) The hypolimnion (50-120 m) harbors peak biodiversity (Shannon H↑), biomass (contig counts↑), and co-occurring resistomes (sul1-intI1; Mantel r = 0.85). Metagenomic functional profiling further reveals dominance of carbohydrate (35%) and amino acid metabolism (43%), with central hypolimnetic communities uniquely enriched in CE11 carbohydrate esterases driving carbon mineralization. We propose stratified interventions: (1) enhanced wastewater treatment/wetlands to curtail nitrogen-driven genomic expansion in northern zones; (2) oxygen nano-bubble injection to disrupt archaeal P-regeneration in profundal layers. This study redefines deep lakes as microbial evolutionary theaters and establishes a framework for ecosystem-sensitive restoration.