Knowledge of how biochar impacts soil denitrification kinetics as well as the mechanisms of interactions is essential in order to better predict the nitrous oxide (N2O) mitigation capacity of biochar additions. This study had multiple experiments in which the effect of three biochar materials produced from corn stover (Zea mays L.), ponderosa pine wood residue (Pinus ponderosa Douglas ex Lawson and C. Lawson), switchgrass (Panicum virgatum L.), and their corresponding biomass materials (corn stover, ponderosa pine wood residue, and switchgrass) on cumulative N2O emissions and total denitrification in soils from two different landscape positions (crest and footslope) were studied under varying water-filled pore space (40, 70, and 90% WFPS). Cumulative N2O emissions were reduced by 30 to 70% in both crest and footslope soils. The effect of biochars and biomass treatments on cumulative N2O emissions and total denitrification were only observed at ≥40% WFPS. The denitrification enzyme activity (DEA) kinetic parameters, K s (half-saturation constant), and V max (maximum DEA rate) were both significantly reduced by biochar treatments, with reductions of 70-80% in footslope soil and 80-90 % in the crest soil. The activation energy (E a) and enthalpy of activation of DEA (ΔH) were both increased with biochar application. The trends in DEA rate constants (K s and V max) were correlated by the trends of thermodynamic parameters (activation energy E a and enthalpy of activation ΔH) for denitrifying enzyme activity (DEA). The rate constant V max/K s evaluated the capacity of biochars to mitigate the denitrification process. Denitrifying enzyme kinetic parameters can be useful in evaluating the ability of biochars to mitigate N2O gas losses from soil.