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We conducted a cross-ecoregion study to test the hypothesis that N-fixation and denitrification would co-occur in streams and rivers across a range of reactive N concentrations. Between 2017 and 2019, we sampled 30 streams in 13 ecoregions, using chambers to quantify N-fixation using acetylene reduction and denitrification using acetylene block. 25 of the study streams were part of the National Ecological Observatory Network or the StreamPULSE network, which provided data on water temperature, light, nutrients, discharge and metabolism. Although N-fixation and denitrification occur under contrasting environmental conditions, we found that they co-occurred in ca. 40% of stream ecosystems surveyed, and microbes capable of carrying out each process were found in all surveyed streams. This dataset includes the chamber data used to calculate nitrogen fixation and denitrification rates, stream substrate information used to scale rates from substrate to whole-reach scale, and a variety of reach-to-landscape scale covariates used to evaluate predictors of rates across the study streams.