Abstract Nitrous oxide (N2O), a potent greenhouse gas, can be generated by compositionally complex microbial populations in diverse contexts. Accurately tracking the dominant biological sources of N2O has the potential to improve our understanding of N2O fluxes from soils as well as inform the diagnosis of human infections. Isotopic Site Preference (SP) values have been used towards this end, as bacterial and fungal nitric oxide reductases produce N2O with different isotopic fingerprints.