Disturbance of wildlife by ecotourism has become a major concern in the last decades. In the Mediterranean, sea-based tourism and related recreational activities are increasing rapidly, especially within marine protected areas (MPAs) hosting emblematic biodiversity. We investigated the impact of ecotourism in the Scandola MPA (UNESCO World Heritage Site, Corsica island), on the population of a conservation flagship, the Osprey Pandion haliaetus. Over the 37-year study period, tourists flow increased sharply. Osprey breeding performance initially increased, but then dropped for pairs nesting within the MPA compared to those breeding elsewhere in Corsica. We examined several hypotheses that could explain such reduction in breeding performance. Recent osprey breeding failures in the MPA are not caused by food scarcity. Using underwater fish surveys, we showed that fish consumed by ospreys were more numerous within the MPA. Focal observation at nests revealed that the overall number of boat passages within 250 m of osprey nests were three times higher inside the MPA compared to a control area. Elevated boat traffic significantly modified osprey time-budgets, by decreasing prey provisioning rate by males, and increasing time spent alarming and flying off the nest in females. This caused stress, and corticosterone levels in chick feathers were three times higher in high-traffic areas compared to places with lower touristic flow in Corsica, the Balearic Islands and Italy. Overall, our integrative, long-term study demonstrates the negative impact of sea-based ecotourism on the Corsican osprey population. This stresses the worldwide importance of rigorously implementing sustainable ecotourism, within well-enforced MPAs.