Removal of excess nitrogen (N) can best be achieved through denitrification processes that transform N in water and terrestrial ecosystems to di-nitrogen (N2) gas. The greenhouse gas nitrous oxide (N2O) is considered an intermediate or end-product in denitrification pathways. Both abiotic and biotic denitrification processes use a single N source to form N2O. However, N2 can be formed from two distinct N sources (known as hybrid N2) through biologically mediated processes of anammox and codenitrification. We questioned if hybrid N2 produced during fungal incubation at neutral pH could be attributed to abiotic nitrosation and if N2O was consumed during N2 formation. Experiments with gas chromatography indicated N2 was formed in the presence of live and dead fungi and in the absence of fungi, while N2O steadily increased. We used isotope pairing techniques and confirmed abiotic production of hybrid N2 under both anoxic and 20% O2 atmosphere conditions. Our findings question the assumptions that (1) N2O is an intermediate required for N2 formation, (2) production of N2 and N2O requires anaerobiosis, and (3) hybrid N2 is evidence of codenitrification and/or anammox. The N cycle framework should include abiotic production of N2.