AbstractN2O is a powerful greenhouse gas contributing both to global warming and ozone depletion. While fungi have been identified as a putative source of N2O, little is known about their production of this greenhouse gas. Here we investigated the N2O-producing ability of a collection of 207 fungal isolates. Seventy strains producing N2O in pure culture were identified. They were mostly species from the orderHypocrealesorder—particularlyFusarium oxysporumandTrichodermaspp.—and to a lesser extent species from the ordersEurotiales, Sordariales and Chaetosphaeriales.The N2O15N site preference (SP) values of the fungal strains ranged from 15.8‰ to 36.7‰ and we observed a significant taxa effect, withPenicilliumstrains displaying lower SP values than the other fungal genera. Inoculation of 15 N2O-producing strains into pre-sterilized arable, forest and grassland soils confirmed the ability of the strains to produce N2O in soil with a significant strain-by-soil effect. The copper-containing nitrite reductase gene (nirK) was amplified from 45 N2O-producing strains and its genetic variability showed a strong congruence with theITSphylogeny, indicating vertical inheritance of this trait. Taken together, this comprehensive set of findings should enhance our knowledge of fungi as a source of N2O in the environment.