Darwin Harbour is a working port and the most populated city in the Northern Territory of Australia. This macrotidal estuary is located in the wet–dry tropics of Northern Australia and notwithstanding mounting development pressures in the region, is largely unmodified. The prevailing oligotrophic condition of estuarine waters suggest that biogeochemical cycling in sediments remain active, buffering the influence of anthropogenic inputs. We tested the hypothesis that nutrient hotspots exist in depositional low-velocity zones, with a gradient of high to low nitrogen processing from the upper to outer reaches of the estuary. A number of factors were examined for their influence on the effectiveness of denitrification in these depositional zones, a putative key process driving nitrogen removal, with particular emphasis on carbon-loading extremes in tidal creeks, spatial gradients along the estuary and the influence of seasonality. There were significant differences in process rates between hypereutrophic/eutrophic tidal creeks that receive the largest proportion of treated sewage loads in the region and the mesotrophic/oligotrophic tidal creeks that were comparatively undisturbed. Net benthic nutrient fluxes and dinitrogen efflux rates were more than an order of magnitude higher and lower, respectively, in degraded (hypereutrophic/eutrophic) tidal creek systems where denitrification efficiency (DE%) was < 40%. Denitrification (Dinitrogen efflux) rates in tidal creeks (mesotrophic/oligotrophic) and broader estuarine sites were high (~ 8 mmol N m−2 day−1) and denitrification efficiency remained > 65%, particularly during the wet season. On a whole-of-estuary basis, denitrification in conjunction with mechanisms such as burial could feasibly make a substantial impact, abating the influence of anthropogenic inputs. Although considerable variability was encountered, particularly across seasons, the hypothesis of elevated denitrification rates as nutrient hotspots in depositional zones along the estuary was not convincing. More influential are tidal creeks as potential ‘reactors’ for N cycling and removal, but their capacity can be degraded by overloading with nutrients.