Marine mammal foraging grounds are popular focal points for marine protected area (MPA) implementation, but may be temporally dynamic, requiring continuous monitoring to infer prey availability and abundance. Marine mammal distributions are assumed to be driven by their prey in foraging areas, but limited understanding of prey distributions often prevents us from exploring how shifting prey availability impacts both seasonal and long-term marine mammal distributions. Environmental DNA (eDNA) metabarcoding could enhance understanding of marine mammal habitat use in relation to their prey through simultaneous monitoring of both. However, eDNA applications focused on marine mammals or predator-prey dynamics have been limited to date. In this study, we assess spatiotemporal changes in the availability and abundance of minke whale (Balaenoptera acutorostrata) prey species in a newly established MPA, employing eDNA metabarcoding. We recovered 105 molecular operational taxonomic units (OTUs) from marine vertebrates using two primer sets targeting 12S and 16S genes, along with 112 OTUs from a broader eukaryotic primer set targeting 18S rRNA. Overall, key forage fish prey species, sandeels and clupeids, were the most abundant teleost fishes detected, although their availability varied temporally and with distance from shore. We also found clear spatial partitioning between coastal bottlenose dolphins and the more pelagic minke whales and harbour porpoises, paralleling availability of their main prey species. Other species of conservation interest were also detected including the critically endangered European eel (Anguilla anguilla), blue fin tuna (Thunnus thynnus), and the invasive pink salmon (Oncorhynchus gorbuscha). This study demonstrates the application of eDNA to detect spatiotemporal trends in the occurrence and abundance of cetacean predators and their prey, furthering our understanding of fine-scale habitat use within MPAs. Future, long-term monitoring of predator-prey dynamics with eDNA could improve our ability to predict climate-induced shifts in foraging grounds and enhance rapid responses with appropriate management actions.