Salt marsh sediments are hotspots for the production of nitrous oxide, an ozone-depleting greenhouse gas. While processes mediated by bacteria and archaea, including denitrification and nitrification, contribute to nitrous oxide production, little is known about the potential contribution of fungi to nitrous oxide production from salt marsh sediments. We have isolated four nitrous oxide-producing fungal strains from the North Inlet salt marshes in South Carolina. We demonstrate that up to 17% of nitrite are converted to nitrous oxide by salt marsh sediment fungi. The site preference (SP) of nitrous oxide produced by salt marsh sediment fungi ranged from 7.5 ± 1.6‰ to 33.4 ± 1.2‰, which was lower than SP reported for the model fungal denitrifier Fusarium oxysporum (37.1 ± 2.5‰) isolated from soil. Metabarcoding of the microbial eukaryotic community in salt marsh sediments targeting the large subunit of the ribosomal RNA gene showed that the genera represented by our fungal strains (Purpureocillium, Trichoderma, and Rhodotorula) were prevalent in the sediments of North Inlet salt marshes. The nitrous oxide SP values we measured will serve as representative endmembers used in isotope mass balance calculations to determine the fungal contribution to nitrous oxide production from salt marsh sediments.