Estuaries are critical to the life histories and development of many marine species, and support some of the most productive aquatic ecosystems. However, owing to their geographic accessibility, many estuaries face considerable anthropogenic threats, which can be exacerbated in developing nations. Understanding the ecological implications of anthropogenic activities as a precursor to dedicated conservation activities requires comprehensive assessments of baseline trophic relationships. This study addresses a shortfall in such information for two tropical, coastal ecosystems globally identified as priority areas for biodiversity conservation: the Paranagua estuarine complex; and the Cananeia-Iguape estuarine lagoon system in southern Brazil. Ecological relationships were investigated through carbon and nitrogen stable isotope (delta C-13 and delta N-15) determination in sediments, plant fragments and the muscle tissues of marine species occupying different trophic levels, including benthic invertebrates, ichthyofauna (Stellifer rastrifer, Paralonchurus brasiliensis, and Isopisthus parvipinnis), and delphinids (Sotalia guianensis and Pontoporia blainvillei). Isotope ratios facilitated distinguishing plant fragments, sediments (which were terrestrially influenced) and biota (predominantly marine influenced). However, despite variation among anthropogenic stressors, isotope ratios did not vary between the studied estuaries, implying similar impacts on trophic relationships which might reflect some inter-estuary mixing. All ichthyofauna and the delphinids had similar generalist diets and shared the same trophic niche, which may reflect their consistent spatio-temporal distributions and habitat patterns in the estuaries. The data illustrate the utility of isotopically characterizing biodiversity-hotspot estuaries as a mechanism for rapidly assessing ecological relationships between key species and regional habitats. Such an approach can be used to evaluate ecological connectivity as a baseline for management and might be applied more broadly, embracing multiple regions as part of estuarine and coastal biome conservation efforts