Abstract: Nitrous oxide (N₂O) is a major greenhouse gas. Estimates suggest that soil-derived N₂O emissions contribute 80% to 90% of the total N₂O released from the entire ecosystem, with farmland soils receiving excessive nitrogen fertilizer exhibiting particularly high N₂O emission intensities. Soil microbial-mediated nitrification and denitrification processes are the primary pathways for N₂O production, and microbial community structure is closely associated with the emission patterns of N₂O and dinitrogen (N₂). Consequently, elucidating the effects of nitrogen fertilizers on soil microbial communities and investigating the microbial mechanisms governing N₂O and N₂ emissions provides a critical theoretical foundation for regulating gaseous nitrogen emissions and enhancing nitrogen fertilizer use efficiency. In this study, quantitative polymerase chain reaction (qPCR) and high-throughput sequencing technology were used to quantify the annual variations in the abundance of key nitrification and denitrification functional genes, as well as the changes in bacterial community structure and diversity, in farmland soils under long-term gradient nitrogen fertilizer application. These measured variables were further analyzed for correlation with soil N₂O and N₂ emission fluxes to explore the relationship between bacteria communities and gaseous nitrogen emissions. The results demonstrated that long-term nitrogen fertilizer application modified soil bacteria community structure and decreased bacterial diversity. Meanwhile, the abundance of certain bacteria with nitrification and denitrification functions increased, which may be associated with the elevated N₂O and N₂ emission fluxes. The annual average abundances of soil AOB amoA, nirK, nirS, and nosZ genes in the N600 treatment were approximately 14.43, 1.16, 1.00, and 1.34 times higher than those in the N0 treatment, respectively. This finding indicates that nitrogen fertilizer application exerts a more pronounced impact on the nitrification functional genes than on denitrification functional genes.