The structure and functioning of ecosystems are largely determined by the interactions between species within a biological community. Among these interactions, species exhibiting similar vertical and spatial prey preferences can be identified, thereby belonging to the same trophic guild. Our study explored some trophic characteristics of a diverse megafaunal community (cetaceans, tunas, seabirds) in the Bay of Biscay (BoB). Using stable isotope analysis (SIA), we explored the dietary habits and niche overlap among predators. The degree of isotopic niche overlap was generally low, but with certain species exhibiting large and narrow isotopic niche areas (long-finned pilot whales and Balearic shearwaters, respectively). Our results revealed a diversity of dietary preferences leading to the identification of three distinct trophic guilds based on prey functional groups and spatial preferences: cephalopod feeders (e.g. long-finned pilot whales, Cuvier’s beaked whales, striped dolphins), crustacean feeders (e.g. fin whales, albacores), and piscivores (e.g. common dolphins, harbour porpoises, bottlenose dolphins, Atlantic bluefin tunas, Balearic shearwaters). Our findings showed resource partitioning and niche differentiation among the megafaunal community, highlighting the complexity of BoB’s marine ecosystem. The insights derived from this study hold important implications for ecosystem management and the implementation of conservation initiatives.