Ecuador's Galapagos Islands and their unique biodiversity are a global conservation priority. We explored the presence, composition and environmental drivers of plastic contamination across the marine ecosystem at an island scale, investigated uptake in marine invertebrates and designed a systematic priority scoring analysis to identify the most vulnerable vertebrate species. Beach contamination varied by site (macroplastic 0–0.66 items·m−2, microplastics 0–448.8 particles·m−2 or 0–74.6 particles·kg−1), with high plastic accumulation on east-facing beaches that are influenced by the Humboldt Current. Local littering and waste management leakages accounted for just 2% of macroplastic. Microplastics (including anthropogenic cellulosics) were ubiquitous but in low concentrations in benthic sediments (6.7–86.7 particles·kg−1) and surface seawater (0.04–0.89 particles·m−3), with elevated concentrations in the harbour suggesting some local input. Microplastics were present in all seven marine invertebrate species examined, found in 52% of individuals (n = 123) confirming uptake of microplastics in the Galapagos marine food web. Priority scoring analysis combining species distribution information, IUCN Red List conservation status and literature evidence of harm from entanglement and ingestion of plastics in similar species identified 27 marine vertebrates in need of urgent, targeted monitoring and mitigation including pinnipeds, seabirds, turtles and sharks. Display omitted •Plastic contamination was identified in all marine habitats surveyed in San Cristobal.•Hotspots for beach plastics are on the eastern coast, up to 449 particles m−2.•Elevated microplastics in surface seawater around the harbour shows local inputs.•Microplastics were found in 52% of marine invertebrates sampled (n = 123).•27 marine vertebrates scored at high risk of harm from entanglement and ingestion.