Further understanding is needed regarding how biochar, over the long term, influences N₂O release and the associated communities of nitrifiers and denitrifiers in paddy soils. This field study examined the responses of these microbial communities to biochar applied for different durations (2016 or 2023) and at different doses (15 or 45 t·ha⁻¹), alongside a control (CK) without biochar addition. Relative to the control (CK), all biochar amendments led to a comprehensive enhancement of soil physicochemical properties. However, their impacts on N₂O fluxes diverged: cumulative emissions rose by 18.44% under the high-rate (45 t·ha⁻¹), first-year application (NB45) in 2023, but were suppressed across all other biochar treatments. Microbial community composition diverged markedly between treatment chambers, with the abundances of Nitrospira and Chloroflexota showing distinct patterns. In 2016, the two bacterial species exhibited significantly high abundance proportions, with maximum shares of 23.55% (2016, 45 t·ha⁻¹) and 12.16% (2016, 45 t·ha⁻¹), the most abundant in nitrification and denitrification, respectively, which influenced the certainty of changes in the microbial community structure. Biochar enhances nitrogen metabolism in nitrifying microorganisms but inhibits denitrification processes, with the biochar applied in 2023 having a remarkable effect. Overall, biochar application effectively enhances soil physicochemical properties, mitigates N₂O emissions over the long term, and modulates the community structure and functional traits of nitrifying and denitrifying microorganisms. These combined effects contribute to promoting environmental security for sustainable development within agricultural production systems while reducing the carbon footprint.