River discharges are usually gauged at sites farther upstream than estuarine tidal reaches. As a result, global estimates of river water and nutrient fluxes to the ocean are likely underestimated as they often neglect groundwater discharge occurring in estuaries downstream of river gauging stations. We used radon and radium isotopes as tracers of groundwater discharge into the Sebastian River Estuary, a gaining stream in Florida, USA. We developed a spatially-distributed mass balance model that accounts for radon sources and sinks in waters above and below the estuarine pycnocline. Radium isotopes ( 224Ra, 223Ra, and 226Ra) were not enriched in groundwater relative to surface water and thus had limited usefulness as tracers at this specific site. The detection of fresh groundwater just beneath the sediment:water interface overlain by brackish bottom water implies that fresh groundwater dominates over saline groundwater in this salt wedge estuary. Lateral groundwater inputs from sandy banks into waters above the estuarine pycnocline were about 6-fold higher than inputs into waters below the pycnocline. Groundwater discharge rates into the surface layer of the estuary estimated from a radon mass balance ranged from 5 to 18 m 3/s (or 18 to 62 cm/day if uniformly distributed throughout the entire estuary area). The fluxes into the bottom layer ranged from 0.8 to 1.1 m 3/s (or 2.8 to 3.9 cm/day). These groundwater inputs augmented river discharges gauged upstream of the estuary tidal reaches by about 260% during the dry period and 135% during the wet period. As nutrient and other dissolved species are often highly enriched in groundwaters, groundwater probably controls surface water quality in this and other Florida estuaries.