ABSTRACT: Knowledge of mesopredator habitat use and trophic ecology is crucial for identifying essential habitat and understanding community and food web dynamics. Defining functional roles of resting elasmobranchs is especially important as they can influence benthic community dynamics by residing in an area for a considerable amount of time. This study combines acoustic telemetry, bulk stable isotope analysis of δ13C and δ15N, and carbon isotope analysis of essential amino acids (δ13CEAA) to investigate the habitat use and trophic ecology of Atlantic stingrays Hypanus sabinus within a subtropical estuary. Diel period, tide height (m), moon illumination, and water temperature (°C) significantly influenced hourly presence of stingrays. Stingray presence increased in water temperatures ranging from 15 to 25°C, but they were more likely to be absent when temperatures were <10 and >30°C. Bulk δ13C and δ15N from stingray tissues exhibited spatial gradients throughout the estuary, likely due to isotopic variability in baseline resource channels (e.g. macroalgae) and dietary differences between individual stingrays. Linear discriminant analysis of δ13CEAA values indicated trophic assimilation of macroalgal essential amino acids, demonstrating the observed spatial gradient in bulk δ13C values may be driven by differential 13C fractionation in macroalgae across space due to environmental conditions, or potentially different species of macroalgae throughout the estuary. These findings provide key insight into the relationship between habitat use and trophic ecology for stingrays, revealing potential responses of vulnerable elasmobranch consumers to future physical and biogeochemical change within estuarine environments.