The Hawkesbury-Nepean River System drains Sydney’s western suburbs and is arguably the most important river system in NSW. In order to build an effective water management plan to accommodate the expected population growth, understanding the sources, delivery pathways and fate of nitrogen both to, and within, the Hawkesbury River is essential. As part of a larger interdisciplinary project, this study focuses on both surface and groundwater pathways for nutrient transport to the tidal freshwater zone. Employing the stable isotope δ15N, the primary objective is to quantify and distinguish nitrogen inputs from groundwater, treated sewage wastewater, agriculture, and natural river processes. Through measuring groundwater nutrient inputs alongside those of the river, a comprehensive understanding of river nutrient dynamics can be achieved. Insights from over a thousand δ15N data points across two seasons reveals distinct isotope ranges across six stations traversing the river. These findings contribute valuable insights into nitrogen sources, transformations and sinks, as well as identifying the potential broader ecological consequences of anthropogenic development along the Hawkesbury River system.