Mercury distribution and bioaccumulation in aquatic ecosystems of tropical Australia is poorly characterised. Barramundi (Lates calcarifer), a widespread high-order predator in both fresh and coastal marine waters of the region, fulfils requirements for a bio-indicator of mercury contamination. In a study of the Mary River system of the Northern Territory, total mercury in the muscle tissue of 300 specimens gathered over four years (2013–2017, across both wet and dry seasons) was determined by direct combustion–atomic absorption spectrometry. Source of nutrition and trophic position of barramundi in the food web was also estimated via carbon and nitrogen isotopes (δ13C and δ15N), respectively, in tissue by stable isotope mass spectrometry, and determination of strontium isotopes (87Sr/86Sr) in otoliths by laser ablation–ICPMS differentiated between freshwater and saltwater residence. Results showed that fish moving into freshwater floodplain wetlands concentrated mercury in muscle tissue at approximately twice the level of those that remained in saline habitats. Resolving life histories through otolith analyses demonstrated diversity in mercury bioaccumulation for individual fish of the same migratory contingent on the floodplains. Although trophic level (δ15N), capture location, source of nutrition (δ13C), and age or size partly predicted mercury concentrations in barramundi, our results suggest that individual variability in diets, migration patterns and potentially metabolism are also influential. Using a migratory fish as a bio-indicator, and tracking its life history and use of resources, proved valuable as a tool to discern hot spots in a coastal waterway for a contaminant, such as mercury.