Per- and polyfluoroalkyl substances (PFAS) are considered as global contaminants of emerging concern in marine ecosystems because of their persistence, bioaccumulation and toxicity. Seabirs are considered excellent indicators for monitoring marine contaminants because they are long-lived, widely distributed, and feed at relatively high trophic levels, allowing the integration of contaminant exposure across space and time. In the present study, we monitored PFAS contamination in the Mediterranean Sea using the Scopoli’s shearwater (Calonectris diomedea), an endemic seabird to the Mediterranean basin, as an ecological indicator. Specifically, we measured blood concentrations of 21 PFAS in Scopoli’s shearwater fledgings. Blood samples (n = 106) were collected from fledglings grown in six colonies across the Mediterranean basin and then analyzed for the qualitative and quantitative determination of PFAS. PFAS were detected in all blood samples, with eighteen compounds consistently detected across all colonies. PFOS was the predominant molecule, followed by PFUnDA and PFNA, whereas the remaining ones were detected at lower concentrations. PFAS contamination patterns differed significantly among colonies, with fledglings from the Greek islands exhibiting higher concentration levels and dissimilar profile compared with conspecifics from the other colonies. Our results showed that PFAS contamination is widespread throughout the entire Mediterranean basin, suggesting that these emerging contaminants may represent additional anthropogenic pressure to those already acting on this species of conservation concern, including fisheries bycatch, overfishing, disturbance and predation by invasive species at breeding sites.