Diet of Bald Eagles (Haliaeetus leucocephalus) in the Florida Bay estuary was determined from prey remains from nest sites and used to evaluate differences between two time periods, the 1972 and 1973 breeding seasons compared with 2009 and 2010. Between these two time periods, Florida Bay underwent a well-documented series of ecological changes beginning in the late 1980s, which significantly altered the ecosystem. To examine the hypothesis that ecological changes may have shifted Bald Eagle diets, we compared 571 remains (30 species) collected in 1972/1973 from 21 nest sites, to 419 remains (22 species) collected sites in 2009/2010 from 11 nest sites. Fish made up the majority of prey in 1972/1973 and 2009/2010 (80.7% and 69.5% by number, respectively) and birds were second (15.8% and 29.1%). Hardhead catfish (Arius felis) skulls made up the majority of individual prey remains in both time periods (55% and 54%). Bald Eagles also ate significantly larger catfish in the 1972/1973 seasons, as the mean total skull length was 10% greater and estimated biomass was 35.7% greater. There was no difference in overall prey diversity between the two time periods; however, analysis of similarities showed prey composition differed. Mullet (Mugilidae), jack (Carangidae), Double-crested Cormorant (Phalacrocorax auritus), and Red-breasted Merganser (Mergus serrator) were the species that contributed most to the dissimilarities between time periods. We suggest that Florida Bay's ecological and prey community changes during this time period influenced Bald Eagle diets.