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Applying biochar and 3,4-dimethylpyrazole phosphate (DMPP) is considered to be effective means of mitigating soil nitrous oxide (N2O) emissions. However, their N2O abatement efficiency can be affected by soil properties and associated functional microorganisms. In this study, we investigated the effects of biochar and/or DMPP application on N2O emissions from different agricultural soils (i.e., vegetable, tea, and peach orchards grown for 1 and 7 years) through an incubation experiment. Biochar application significantly reduced N2O emissions from vegetable and tea soils by inhibiting ammonia oxidation. In contrast, in peach orchard soils, adding biochar promoted N2O emissions due to the positive effect of biochar on ammonia-oxidizing bacteria (AOB), which accelerated ammonia oxidation and the inhibition of N2O reduction. DMPP application mitigated 57–74% of soil N2O emissions due to its inhibitory effect on AOB. When biochar and DMPP were co-applied, biochar reduced the efficiency of DMPP in mitigating N2O emissions from tea and peach soils as biochar addition weakened the inhibitory effect of DMPP on AOB. However, N2O emissions from vegetable soils could be further reduced by the synergistic inhibition of ammonia oxidation by biochar and DMPP. Our results suggest that DMPP had a stable mitigation effect on N2O emissions in different soils, while biochar addition could potentially weaken the efficiency of DMPP. The mitigation effect of the co-application of biochar and nitrification inhibitors needs to be explored in future studies under field conditions. [ABSTRACT FROM AUTHOR]