The Estero Bay Watershed, located along the southwest coast of Florida, contains rivers and streams that ultimately discharge into the Gulf of Mexico. This region is influenced not only by rainfall and surface runoff but also by groundwater contributions from the underlying surficial aquifer system. The extent to which groundwater enters surface water bodies, and the potential for it to transport contaminants, remains an important area of investigation. To assess this relationship, water samples were collected from 22 locations over a six-month period during Florida’s summer rainy season. Groundwater and surface water samples were analyzed for naturally occurring tracers, including temperature, salinity, stable isotopes of hydrogen and oxygen, and short-lived radium isotopes. These tracers were selected to distinguish between groundwater and surface water sources. Groundwater was observed to be cooler and less saline than stream water, while isotopic compositions and radium activity levels varied by location. These differences are being used to inform mixing models that estimate the proportion of groundwater contributing to streamflow. Findings from this study are expected to enhance understanding of groundwater–surface water interactions in coastal watersheds and to support resource managers in efforts to protect water quality in the Estero Bay region.