Darwin Harbour is a macrotidal estuary located in the wet–dry tropics of Northern Australia. This largely unmodified oligotrophic harbour is also a working port, where the largest proportion of the Northern Territory population reside and future industrial and urban development are planned. A contemporary examination of nitrogen sources and loading is presented to aid further evaluation of this pressure and infer some of important mechanisms that drive the nutrient status of this estuarine system. Nitrogen inputs as total nitrogen (TN) from watersheds and point-sources, predominantly wastewater treatment plants (WwTP), were modelled for 2007 and 2017 to provide a decadal evaluation of N loads in alignment with the generation of revised regional land use. The model was applied to 21 sub-catchments that have been subject to expanding urbanisation since 2007. Nitrogen yields ranged from 3 kg N ha/y to 23 kg N ha/y increasing with the proportion of urbanisation in the catchment. Comparison between 2007 and 2017 showed that riverine, watershed and point-source inputs had increased by 40% coinciding with urban expansion. Wet season rainfall was a primary means of nitrogen transport, and variability of climate and land use interact to amplify riverine N exports, with urbanisation an important agent of change influencing the contribution of terrestrially derived sources of nitrogen. The last of which contributed approximately 5%–10% of N inputs to the estuary, but was outweighed by oceanic sources (>90%). Current water quality condition and nitrogen limitation of estuarine waters suggests mechanisms, such as watershed nitrogen retention and biogeochemical cycling, particularly denitrification, in receiving waters and sediments remain active despite oceanic inputs and increasing anthropogenic loads.