Among strategies suggested to decrease agricultural soil N2O losses, the use of nitrification inhibitors such as DMPP (3,4-dimethylpyrazole phosphate) has been proposed. However, the efficiency of DMPP might be affected by soil amendments, such as biochar, which has been shown to reduce N2O emissions. This study evaluated the synergic effect of a woody biochar applied with DMPP on soil N2O emissions. A incubation study was conducted with a silt loam soil and a biochar obtained from Pinus taeda at 500 °C. Two biochar rates (0 and 2% (w/w)) and three different nitrogen treatments (unfertilized, fertilized and fertilized + DMPP) were assayed under two contrasting soil water content levels (40% and 80% of water filled pore space (WFPS)) over a 163 day incubation period. Results showed that DMPP reduced N2O emissions by reducing ammonia-oxidizing bacteria (AOB) populations and promoting the last step of denitrification (measured by the ratio nosZI + nosZII/nirS + nirK genes). Biochar mitigated N2O emissions only at 40% WFPS due to a reduction in AOB population. However, when DMPP was applied to the biochar amended soil, a counteracting effect was observed, since the N2O mitigation induced by DMPP was lower than in control soil, demonstrating that this biochar diminishes the efficiency of the DMPP both at low and high soil water contents.