Fishermans Bend is an urban redevelopment precinct situated on the Yarra River estuary in Melbourne, Australia. Understanding the hydrogeological system is important for characterising the impacts from legacy contamination and for monitoring the effects of urbanisation on groundwater flow systems and quality. Stable isotopes of water (δ18O, δ2H) and carbon (δ13C), radioisotopes (3H, 14C) and other geochemical indicators were used to assess sources of water and salinity in the shallow groundwater. Groundwater in the upper aquifer was predominantly Ca-HCO3− dominant, with fresh to brackish salinity—189–3,680 mg/L total dissolved solids (TDS). Localised areas of Ca-SO42− and Na-HCO3− dominant groundwater were impacted by industrial activities and legacy landfills, respectively. Stable isotopes (e.g. δ18O −5.7 to −2.9‰) and tritium activities (1.75–2.45 TU) within the aquifer indicate meteoric water recharged by modern rainfall with short residence times. Carbonate dissolution from shell material, and decay of organic waste and methanogenesis in landfill-leachate-impacted bores were shown to enrich δ13C values up to −4.2‰. In contrast, groundwater in the adjacent/lower aquitard was Na-Cl dominant and saline (19,600–23,900 mg/L TDS), with molar ratios reflective of ocean water, indicating relict emplaced salts. This is consistent with 14C dating of shell material, indicating deposition in a Holocene marine environment. The presence of tritium above background levels (0.20–0.35 TU) in the groundwater, however, suggests a component of modern recharge. Salinity fluctuations within the aquitard at times of peak river level suggest the modern water source is ingress from the adjacent Yarra River.