Rapid population growth and land-use intensification over the last century have resulted in a substantial increase in nutrient loads degrading marine and freshwater ecosystems worldwide. In mixed-use watersheds, elevated nitrogen loads from wastewater treatment plant (WWTP) effluent or agricultural runoff often drive the eutrophication of waterways. Accordingly, the objective of this research was to identify sources of riverine nitrate (NO3), a deleterious dissolved species of nitrogen, with a combined isotopic tracing technique in the Bow River and the Oldman River in Alberta, Canada. Riverine NO3 and boron (B) concentrations, mean daily flux and δ15NNO3, δ18ONO3, and δ11B values were determined at 17 mainstem sites during high and low discharge periods in 2014 and 2015. The data for mainstem sites were then compared to results for effluent from seven WWTPs, eight synthetic fertilizers, cow manure, and three predominantly agricultural tributary sites to estimate point and non-point NO3 sources. The NO3 flux, δ15NNO3 and δ18ONO3 values indicated the city of Calgary’s Bonnybrook WWTP effluent accounts for the majority of the NO3 flux in the Bow River downstream of Calgary. δ15NNO3 and δ11B values in the Bow River highlighted an increase in agricultural NO3 loading downstream of irrigation return-flows. A three-fold decrease in the NO3:B flux ratio indicated NO3-removal processes are active in the lower reaches of the Bow River. For the Oldman River, δ11B values revealed elevated nutrient loading from the Lethbridge WWTP effluent (10% of downstream B flux). Furthermore, the agricultural tributaries contributed 25% of the local B flux to the Oldman River. Overall, δ11B was proven to be an effective co-tracer for discriminating between urban and agricultural sources of NO3 in these large mixed-use watersheds. This combined isotope tracing approach has significant potential to identify point and non-point NO3 sources driving eutrophication around the world.