In order to study the pollution of an aquatic ecosystem, it is necessary to analyze not only the levels of chemical pollutants in water, but also those accumulated in the sediment matrix, as well as to assess its ecotoxicological status. The Alqueva reservoir, the largest artificial lake in Europe, was chosen as case study as it constitutes the most important water supply source in southern Portugal. It is located in the Guadiana River Basin, in a semi-arid region with high levels of water scarcity and where agriculture is one of the main activities. The evaluation of sediments comprised: (1) physical and chemical analysis (grain size, pH, organic matter, nitrogen, phosphorus); (2) potentially toxic trace elements (Cu, As, Pb, Cr, Cd, Zn and Ni); and (3) ecotoxicological evaluation with Vibrio fischeri, Thamnocephalus platyurus, Daphnia magna, and Heterocypris incongruens. Total trace element concentrations indicated that As, Cd, and Pb surpassed the Canadian levels for the protection of aquatic life, in most of Alqueva's sites. The results of the toxicity assessment showed that some locations induced acute and chronic toxicity in the species used. Further, the H. incongruens was the most sensitive species as far as the contamination found in the sediment is concerned, followed by the bacteria V. fischeri. This integrative approach, together with the water column quality assessment, allowed a comprehensive evaluation of the environmental quality of this strongly modified water body and will allow the implementation of remediation strategies to obtain a good ecological potential as proposed in the Water Framework Directive.