Marine turtles are sensitive to harvesting because of life-cycle traits such as longevity, late maturity and natal philopatry. The take of nesting females is of conservation concern because they are key to population maintenance and has led to global efforts to protect this life stage. In the Turks and Caicos Islands (TCI; a UK Overseas Territory in the Caribbean), previous turtle fishery legislation protected nesting turtles on the beach but not in the water, where turtles over a minimum size were subject to legal take. In a 2-year study, we undertook nesting beach and in-water surveys, molecular analyses, satellite tracking and collation of fisheries landing data to investigate the populations of green (Chelonia mydas) and hawksbill (Eretmochelys imbricata) turtles in the TCI and its turtle fishery. Adults were frequently taken in one of the region’s largest legal and artisanal turtle fisheries. We suggest that nesting populations in the TCI, which contain genetically unique haplotypes, have diminished since the 1980s, likely as a result of the harvest of adults. Using these multiple lines of evidence, we highlight the inadequacies of the former fishery regulations and propose specific legislative amendments, which, as a result of this study, were implemented on 1 July 2014 by the TCI government. With good enforcement, these measures will protect adults breeding in the TCI and those from nesting rookeries in the region that use the waters of the TCI, improve the management of this fishery, and safeguard fisher livelihoods.