The global pool of reactive nitrogen has doubled in the last century in response to the need to increase food production with the consequent increase in fertiliser-derived reactive nitrogen detrimentally affecting aquatic ecosystems. This study investigates the spatial distribution and significance of denitrification in the lowland, agriculturally-impacted River Wensum catchment in eastern England as a natural attenuation process. To investigate the evidence for denitrification, the hydrochemical characteristics and dual stable isotope composition of nitrate (15N and 18O) were measured over a 15-month period, 2015–2017, in 63 samples of field drainage in predominantly clay loam and sandy clay loam soils under mainly arable cultivation. Microbially-mediated denitrification in field drainage was indicated by the gradient of the linear regression of 15NNO3 and 18ONO3 compositions with a value of 0.58. Dual fractionation of the nitrate isotopes yielded enrichment factors for δ15NNO3 (−4.52‰) and δ18ONO3, (−4.51‰) within the reported ranges for denitrification in aquatic studies. Soil type influenced denitrification, with a positive relationship between percentage clay and δ15NNO3 and δ18ONO3 values. The same relationship was observed for denitrification rates calculated via a simple mass balance approach, which ranged from 11.0 to 26.3 kg N ha−1 and accounted for 30–73% of the leached soil nitrogen. Higher denitrification rates were recorded in drainage areas with a greater soil clay content (>20% by weight). Comparing calculated dentification rates for individual drain areas with median δ15NNO3 values of drain samples demonstrated that an isotopic enrichment of +1‰ is associated with a denitrification rate of 2.6 kg N ha−1. In conclusion, sustainable agricultural practices that maintain natural attenuation processes such as denitrification, for example by preserving and increasing the soil organic carbon content, are desirable to improve overall soil health to support ecosystem services that reduce nitrate pollution.