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Microplastic pollution threatens some of the world's most iconic locations for marine biodiversity, including the remote Galápagos Islands, Ecuador. Using the Galápagos penguin (Spheniscus mendiculus) as a sentinel species, the present study assessed microplastics and suspected anthropogenic cellulose concentrations in surface seawater and zooplankton near Santa Cruz and Galápagos penguin colonies (Floreana, Isabela, Santiago), as well as in penguin potential prey (anchovies, mullets, milkfish) and penguin scat. On average, 0.40 ± 0.32 microplastics L−1 were found in surface seawater (<10 μm; n = 63 L), while 0.003, 0.27, and 5.12 microplastics individual−1 were found in zooplankton (n = 3372), anchovies (n = 11), and mullets (n = 6), respectively. The highest concentration (27 microplastics individual−1) was observed in a single milkfish. Calculations based on microplastics per gram of prey, in a potential diet composition scenario, suggest that the Galápagos penguin may consume 2881 to 9602 microplastics daily from prey. Despite this, no microplastics or cellulose were identified in 3.40 g of guano collected from two penguins. Our study confirms microplastic exposure in the pelagic food web and endangered penguin species within the UNESCO World Heritage site Galápagos Islands, which can be used to inform regional and international policies to mitigate plastic pollution and conserve biodiversity in the global ocean. Environ Toxicol Chem 2024;43:1442–1457. © 2024 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC. [ABSTRACT FROM AUTHOR]