Based on a one-year pilot plant operation of a two-step biofilm nitritation-anammox pilot plant, N2O mitigation strategies were identified by applying a newly developed biofilm modeling approach. Due to adapted plant operation, the N2O emission could be diminished by 75% (8.8% → 2.3% of NH4-Noxidized_AOB). The results (measurement and simulation) confirm the huge importance of denitrification as an N2O source or N2O sink, depending on the boundary conditions. A significant reduction of N2O emissions could only be achieved with a one-step deammonification system, which is related to low nitrite and HNO2 concentrations. Increased oxygen concentrations in the bulk phase are not related to decreased emissions. N2O formation by ammonium-oxidizing bacteria (AOB) just shifts deeper into the biofilm; zones with low oxygen concentrations are not avoidable in biofilm systems. Low oxygen concentrations in the bulk phase, however, result in a reduction of the total net N2O formation due to increased activity of heterotrophic bacteria directly at the source of N2O formation (outer biofilm layer). For the model-based identification of mitigation strategies, the standard modeling approaches for biofilms were expanded by including the factor-based N2O formation and emission approach. The new model 'Biofilm/N2OISAH' was successfully validated using data from pilot-scale measurement campaigns. Altogether, the investigation confirms that the employed digital model can strongly support the development of N2O mitigation strategies without the need for specialized measurement inside the biofilm