Biochar amendment has been shown to reduce nitrous oxide (N2O) emissions from acidic soils in tea plantations. Given that both soil bacterial and fungal denitrifications can produce N2O, their relative contributions to the decrease in N2O emissions following biochar amendment remain unclear. Here, we examined soils sampled from a subtropical tea plantation that had received 2 years of biochar amendment. Measurements of the relative contributions of fungi and bacteria to N2O production were taken by the substrate-induced respiration method implemented with the addition of selective inhibitors. The abundances of total fungi, bacteria, and key N2O-related bacterial genes were quantified by q-PCR, and the composition of the fungal community was analyzed by 18S rRNA amplicon sequencing. The results showed that the contribution of fungi to N2O production (52%) was greater than that of bacteria (18%) for the N-applied acidic soils. Biochar amendment significantly decreased the fungal abundances and the fungal contribution to N2O production (by 28%). In contrast, biochar amendment significantly increased the abundances of N2O-related bacteria (e.g., ammonia-oxidizing bacteria (AOB), nirS, nosZ). Structural equation models (SEMs) revealed that biochar decreased the fungal contribution to N2O production through enhancing the soil pH and shifting the fungal community composition. Our results highlight that the decreased N2O emissions could be ascribed to the stimulated N2O consumption process (driven by N2O-consuming bacteria encoded by the nosZ gene) and suppressed fungal dominance in acidic soils from tea plantations. This study presents relatively comprehensive insights into the regulatory roles of biochar on soil microbe-mediated N2O production processes.