Fungi in terrestrial environments are known to play a key role in denitrification and heterotrophic nitrification. In contrast, the role of fungi in marine nitrogen cycling has remained underexplored and largely neglected, especially in the open ocean. We coupled 15N-tracer experiments with a selective inhibition method to determine the contribution of marine fungi to N2O production in the eastern tropical North Pacific oxygen minimum zone. Fungal community composition evaluated by the internal transcribed spacer region 2 (ITS2) revealed that taxa from the phyla Basidiomycota and Ascomycota dominated most depths.