Aims <p>Anthropogenic nitrogen (N) input increases both soil N availability and acidity; however, the responses of soil microbes to these changes and their impacts on soil carbon (C) cycling under long-term excessive N fertilization remain unclear.</p> Methods <p>A 15-year N gradient experiment was conducted in southwest China, comprising four N application levels: 0, 285, 480, and 600&#xa0;kg N ha⁻¹&#xa0;year⁻¹. A suite of metrics was employed, including amino sugars, lignin phenols, microbial C use efficiency, C-degradation enzyme activities, ¹³C-nuclear Magnetic resonance spectroscopy, 16S rRNA and ITS sequencing, and high-throughput quantitative PCR.</p> Results <p>Nitrogen fertilization substantially increased soil N availability (elevating nitrate and ammonium concentrations) and acidified the soil (lowering soil pH). Variance partitioning analysis indicated that both bacterial and fungal communities were influenced by the interaction between N availability and soil acidification. Fungal communities primarily responded to elevated N availability by enriching nitrophilic copiotrophs (e.g., <i>Eurotiales</i>), whereas bacterial communities mainly reacted to soil acidification by enriching acid-tolerant copiotrophs (e.g., <i>Actinobacteria</i>). These microbial shifts led to a 113.9%–196.7% increase in bacterial necromass, and its contribution to soil organic carbon (SOC) rose by 105.1%–133.1%. Additionally, the enrichment of fungal nitrophilic copiotrophs reduced the abundance of lignin-degrading genes (e.g., <i>glx</i>) and C-oxidase activities. Consequently, Lignin phenols increased by 97.3%–112.2%, and plant-derived C contributions to SOC rose by 25.2%–58.5%.</p> Conclusions <p>This study provides valuable insights into how microbial communities adapt to soil acidification and N enrichment following long-term N fertilization, and how such adaptations enhance SOC sequestration.</p>