By measuring the trophic niche (the diversity of dietary resources used by a species), we can understand how species use and depend on dietary resources. This is especially important in elasmobranchs, given their steep declines and important roles as predators that shape communities and the flow of biomass through ecosystems. Using the niche concept, we aimed to compare an assemblage of euryhaline and coastal elasmobranchs in the South Alligator River (Carcharhinus leucas, C. amboinensis, Himantura dalyensis, Glyphis garricki, G. glyphis, P. pristis and Rhizoprionodon taylori) using stable isotopes (SI) and fatty acids (FA) extracted from muscle tissue samples. We used SI Bayesian ellipses to calculate niche space and overlap among species, with sufficient sample numbers and a modified version of this to calculate FA niche metrics. We compared species δ13C values and found two guilds of species: one with a marine signature, and the other with an estuarine/freshwater base. Fatty acids largely corroborated these results. However, there were slight differences, particularly for C. leucas, which may have been caused by temporal differences in the uptake of biomarkers. Carcharhinus leucas had the largest SI niche space and was overlapped by R. taylori. Overall, we found that these species provide important connections across riverine, estuarine and coastal ecosystems.