Here we provide the first demonstration of the potential for N2O production by soil-isolated nitrate-ammonifying bacteria under different C and N availabilities, building on characterizations informed from model strains. The potential for soil-isolated Bacillus sp. and Citrobacter sp. to reduce NO3-, and produce NH4+, NO2- and N2O was examined in batch and continuous (chemostat) cultures under different C-to-NO3- ratios, NO3--limiting (5 mM) and NO3--sufficient (22 mM) conditions. C-to-NO3- ratio had a major influence on the products of nitrate ammonification, with NO2-, rather than NH4+, being the major product at low C-to-NO3- ratios in batch cultures. N2O production was maximum and accompanied by high NO2- production under C-limitation/NO3-sufficiency conditions in chemostat cultures. In media with lower C-to-NO3-N ratios (5- and 10-to-1) up to 2.7% or 5.0% of NO3- was reduced to N2O by Bacillus sp. and Citrobacter sp., respectively, but these reduction efficiencies were only 0.1% or 0.7% at higher C-to-NO3- ratios (25- and 50-to-1). As the highest N2O production did not occur under the same C-to-NO3- conditions as highest NH4+ production we suggest that a re-evaluation may be necessary of the environmental conditions under which nitrate ammonification contributes to N2O emission from soil.