Nutrient pollution in waterways is one of the most widespread, costly and challenging environmental problems in the world. Increasing nutrient loads, among other stressors, are challenging the water quality of the River Derwent and its estuary. Recent years have seen summer-time blue green algal blooms in the river and upper estuary. One hypothesis is that elevated levels of nitrogen (N) have stimulated algal growth in the estuary. The estuary contains many sources of N, including wastewater treatment plant effluent, fish farm effluent, dairy farm manure, fertilizer runoff, pulp mill effluent, as well as stormwater runoff. These sources may be identified and quantified using isotopic fingerprinting of nitrogen, provided that source signatures are isotopically distinct. Previous research has shown that N isotopic fingerprints in macroalgae are useful bioindicators. In addition, sediment cores may provide information about the history of nutrient loads and sources. In this study, common ulvoids (e.g. Ulva australis) along rocky reefs of the estuary foreshore and sediment cores across the estuary were sampled and analysed for their N content and isotopic composition. Results show that sampled ulvoids in the lower and middle estuary predominantly use a mixture of two N sources, marine N and sewage N from waste water treatment plants. Sewage N contributions range from <1% near Tinderbox to 100% at Cameron Bay. Results from sediment cores show that sediment N in the middle estuary is dominated by an isotopic signature representative of pulp mill effluent. Elevated N isotopic values of sediment from the lower estuary are indicative of marine N sources, but the total nitrogen content has increased since 1970. Our study demonstrates that nitrogen isotopic signatures in this estuary are useful to differentiate between sewage, marine, and terrestrial sources.