Nitrous oxide (N2O) is one of the gases with the greatest impact in the atmosphere due to its persistence and significant contribution to the greenhouse effect. This study provides an insight into the dynamics of N2O production in wastewater nitrogen removal systems. A 10 L sequencing batch reactor containing enriched anammox biomass was subjected to different operational conditions, i.e., temperature, feed time, NO2−/NH4+ ratio and the initial concentrations of NH4+ and NO2−. Tests showed no significant differences in maximum N2O production when the system was operated with a shorter feed time and no increase in the operating temperature. A higher N2O production was observed when the initial NO2−/NH4+ ratio increased from 1.3 to 1.7 and 1.9. The highest initial concentration of NO2− was linked to an increase in residual N2O at the end of the batch cycle, probably due to heterotrophic denitrifying metabolism.