Nitrous oxide (N2O) production by filamentous fungi has been demonstrated in pure culture and has been estimated indirectly in soils. However, it is unknown whether ectomycorrhizal fungi can also produce N2O. We demonstrate for the first time the ability of nitrogen (N)-tolerant ectomycorrhizal fungi (Paxillus involutus and Tylospora fibrillosa), found in forest soils under moderate to high rates of N deposition, to produce N2O from nitrate reduction. The N2O concentrations from the ectomycorrhizal fungal treatments after a 10-day pure culture experiment were 0.0117±0.00015 (P. involutus) and 0.0114±0.0003 (T. fibrillosa), and 0.0114±0.00043 μmol N2O L−1 from a known fungal denitrifier (Fusarium lichenicola). No N2O was detected in the control treatment. Our results indicate the potential for these two N-tolerant ectomycorrhizal fungi to contribute to N2O production. Given that these species are abundant in many forest soils, the strength and regulation of fungal N2O production should now be verified in situ.