Understanding the foraging ecology of endangered marine mammals is crucial for their conservation yet remains challenging due to the inaccessibility of their feeding habitats. Here, we used environmental DNA (eDNA) metabarcoding with two complementary 12S rRNA markers to characterize potential prey communities available to the critically endangered Rice’s whale (Balaenoptera ricei) in its core habitat in the northeastern Gulf of America (formerly Gulf of Mexico). Water samples collected at 21 stations during a 2019 survey detected 99 unique fish species across 62 families, exceeding the diversity recorded by concurrent trawl surveys. To support these analyses, existing reference databases were expanded by sequencing the 12S rRNA gene from 15 regional fish species, using new primers developed for this study. The combined metabarcoding approach revealed 74 fish species not recorded in trawls, while 16 trawl-caught species went undetected by eDNA. Notably, eDNA yielded higher detection rates for several potential prey taxa previously identified through stable isotope analysis and trawl surveys, resulting in an updated list of top potential prey. These findings suggest that key prey species may be more prevalent in Rice’s whale habitat than previously documented. Our study demonstrates the value of eDNA as a complementary tool for monitoring the prey community of this critically endangered cetacean, while highlighting the need for continued development of reference databases to maximize the ecological insights gained from marine metabarcoding applications.