Abstract Background Microbial denitrification is not considered important in human-associated microbial communities. Accordingly, metabolic investigations of the microbial biofilm communities of human dental plaque have focused on aerobic respiration and acid fermentation of carbohydrates, even though it is known that the oral habitat is constantly exposed to nitrate (NO 3 - ) concentrations in the millimolar range and that dental plaque houses bacteria that can reduce this NO 3 - to nitrite (NO 2 - ). Results We show that dental plaque mediates denitrification of NO 3 - to nitric oxide (NO), nitrous oxide (N 2 O), and dinitrogen (N 2 ) using microsensor measurements, 15 N isotopic labelling and molecular detection of denitrification genes. In vivo N 2 O accumulation rates in the mouth depended on the presence of dental plaque and on salivary NO 3 - concentrations. NO and N 2 O production by denitrification occurred under aerobic conditions and was regulated by plaque pH. Conclusions Increases of NO concentrations were in the range of effective concentrations for NO signalling to human host cells and, thus, may locally affect blood flow, signalling between nerves and inflammatory processes in the gum. This is specifically significant for the understanding of periodontal diseases, where NO has been shown to play a key role, but where gingival cells are believed to be the only source of NO. More generally, this study establishes denitrification by human-associated microbial communities as a significant metabolic pathway which, due to concurrent NO formation, provides a basis for symbiotic interactions.