Man-made endocrine-disrupting chemicals (EDCs) range across all continents and oceans. Some geographic areas are potentially more threatened than others: one of these is the Mediterranean Sea. Levels of some xenobiotics are much higher here than in other seas and oceans. In this paper we review the final results of a project supported by the Italian Ministry of the Environment, in which the hypothesis that Mediterranean top predator species (such as large pelagic fish and marine mammals) are potentially at risk due to EDCs was investigated. We illustrate the need to develop and apply sensitive methodological tools, such as biomarkers (Vitellogenin, Zona Radiata proteins and CYP1A activities) for evaluation of toxicological risk in large pelagic fish top predators (Swordfish, (Xiphias gladius), Bluefin Tuna (Thunnus thynnus thynnus)) and nondestructive biomarkers (CYP1A activities and fibroblast cell culture in skin biopsy), for the hazard assessment of threatened marine mammals species (Striped Dolphin, (Stenella coeruleoalba), Bottlenose Dolphin (Tursiops truncatus), Common Dolphin (Delphinus delphis) and Fin Whale (Balaenoptera physalus))exposed to EDCs. Differential gender susceptibility to EDCs is also explored both in large pelagic fish and in cetaceans. In cetaceans, male specimens showed higher cytochrome P450 induction (BPMO in skyn biopsies, CYP2B in fibroblasts cell cultures) by xenobiotics with respect to females.