Due to its high global warming potential, nitrous oxide (N 2O) emissions from wastewater treatment processes have recently received a high degree of attention. Nevertheless, there is still a lack of information regarding the microbiological processes leading to N 2O production. In this study, two lab‐scale sequencing batch reactors were operated with deammonification biomass to investigate the role of denitrification and the influence of substrate availability regarding N 2O formation during the anoxic phase of deammonification. Three different operational phases were established: within the first phase conversion by anammox was favoured and after a transition phase, denitrification activity was promoted. Low nitrous oxide production was observed during stable operation aiming for anammox conversion. Pulsed inflow of the wastewater containing ammonium (NH 4 +) and nitrite (NO 2 ‐) led to increased N 2O production rates. Within the period of denitrification as dominating nitrogen conversion process, the nitrous oxide concentration level was higher during continuous inflow conditions, but the reaction to pulsed inflow was less pronounced. The results indicated that denitrification was responsible for N 2O formation from the deammonification biomass. Operational settings to achieve suppression of denitrification processes to a large extend were deducted from the results of the experiments.