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<p>Nitrous oxide (N<sub>2</sub>O) dominates greenhouse gas emissions in wastewater treatment plants (WWTPs). Formation of N<sub>2</sub>O occurs during biological nitrogen removal, involves multiple microbial pathways, and is typically very dynamic. Consequently, N<sub>2</sub>O mitigation strategies require an improved understanding of nitrogen transformation pathways and their modulating controls. Analyses of the nitrogen (N) and oxygen (O) isotopic composition of N<sub>2</sub>O and its substrates at natural abundance have been shown to provide valuable information on formation and reduction pathways in laboratory settings, but have never been applied to full-scale WWTPs.</p><p>Here we show that N-species isotope ratio measurements at natural abundance level, combined with long-term N<sub>2</sub>O monitoring, allow identification of the N<sub>2</sub>O production pathways in a full-scale plug-flow WWTP (Hofen, Switzerland). The proposed approach can also be applied to other activated sludge systems. Heterotrophic denitrification appears as the main N<sub>2</sub>O production pathway under all tested process conditions, while nitrifier denitrification was less important, and more variable. N<sub>2</sub>O production by hydroxylamine oxidation was not observed. Fractional N<sub>2</sub>O elimination by reduction to dinitrogen (N<sub>2</sub>) during anoxic conditions was clearly indicated by a concomitant increase in SP, δ<sup>18</sup>O(N<sub>2</sub>O) and δ<sup>15</sup>N(N<sub>2</sub>O). The extent of N<sub>2</sub>O reduction correlated with the availability of dissolved inorganic N and organic substrates, which explains the link between diurnal N<sub>2</sub>O emission dynamics and organic substrate fluctuations. Consequently, dosing ammonium-rich reject water under low-organic-substrate conditions is unfavourable, as it is very likely to cause high net N<sub>2</sub>O emissions.</p><p>Our results demonstrate that monitoring of the N<sub>2</sub>O isotopic composition holds a high potential to disentangle N<sub>2</sub>O formation mechanisms in engineered systems, such as full-scale WWTP. Our study serves as a starting point for advanced campaigns in the future combining isotopic technologies in WWTP with complementary approaches, such as mathematical modelling of N<sub>2</sub>O formation or microbial assays to develop efficient N<sub>2</sub>O mitigation strategies.</p>