Nitrogen fertilisation of agricultural soil contributes significantly to emissions of the potent greenhouse gas nitrous oxide (N2O), which is generated during denitrification and, in oxic soils mainly by, ammonia oxidisers. Although laboratory cultures of ammonia oxidising bacteria (AOB) and archaea (AOA) produce N2O, their relative activities in soil are unknown. This work tested the hypothesis that AOB dominate ammonia oxidation and N2O production under conditions of high inorganic ammonia (NH3) input, but result mainly from the activity of AOA when NH3 is derived from mineralisation. 1-octyne, a recently discovered inhibitor of AOB, was used to distinguish N2O production resulting from archaeal and bacterial ammonia oxidation in soil microcosms, and specifically inhibited AOB growth, activity and N2O production. In unamended soils, ammonia oxidation and N2O production were lower and resulted mainly from ammonia oxidation by AOA. The AOA N2O yield relative to nitrite produced was half that of AOB, likely due to additional enzymatic mechanisms in the latter, but ammonia oxidation and N2O production were directly linked in all treatments. Relative contributions of AOA and AOB to N2O production therefore reflect their respective contributions to ammonia oxidation. These results suggest potential mitigation strategies for N2O emissions from fertilised agricultural soils.