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Within oligotrophic ecosystems, resource limitations coupled with interspecific variation in morphology, physiology, and life history traits may lead to niche partitioning among species. How generalist predators partition resources and their mechanisms, however, remain unclear across many ecosystems. We quantified niche partitioning among upper trophic level coastal and estuarine species: American alligators (Alligator mississippiensis), bull sharks (Carcharhinus leucas), common bottlenose dolphins (Tursiops truncatus), common snook (Centropomus undecimalis), and Atlantic tarpon (Megalops atlanticus) in the Shark River Estuary of the Florida Coastal Everglades, USA using acoustic telemetry, stable isotope analysis, and visual surveys, combined with published diet and life history demographic information. Spatial and isotopic niche overlap occurred among most species, with variability in partitioning among interspecific interactions. However, seasonal variability in habitat use, movements patterns, and trophic interactions may promote coexistence within this resource-limited estuary. Beyond guild-level niche partitioning, predators within the Shark River Estuary also exhibit partitioning within species through individual specializations and divergent phenotypes, which may lead to intraspecific variability in niche overlap with other predators. Niche differentiation expressed across multiple organizational levels (i.e., populations and communities) coupled with behavioral plasticity among predators in oligotrophic ecosystems may promote high species diversity despite resource limitations, which may be important when species respond to natural and human-driven environmental change.