Nitrous oxide (N2O) is an unwanted byproduct during biological nitrogen removal processes in wastewater. To establish strategies for N2O mitigation, a better understanding of production mechanisms and their controls is required. A novel stable isotope labeling approach using 15N and 18O was applied to investigate pathways and controls of N2O production by biomass taken from a full-scale nitritation-anammox reactor. The experiments showed that heterotrophic denitrification was a negligible source of N2O under oxic conditions (≥0.2 mg O2 L-1). Both hydroxylamine oxidation and nitrifier denitrification contributed substantially to N2O accumulation across a wide range of conditions with varying concentrations of O2, NH4+, and NO2-. The O2 concentration exerted the strongest control on net N2O production with both production pathways stimulated by low O2, independent of NO2- concentrations. The stimulation of N2O production from hydroxylamine oxidation at low O2 was unexpected and suggests that more than one enzymatic pathway may be involved in this process. N2O production by hydroxylamine oxidation was further stimulated by NH4+, whereas nitrifier denitrification at low O2 levels was stimulated by NO2- at levels as low as 0.2 mM. Our study shows that 15N and 18O isotope labeling is a useful approach for direct quantification of N2O production pathways applicable to diverse environments.