The Guadiana Basin (SW Iberian Peninsula) is affected by acid mine drainage (AMD), a consequence of ancient mining activities in the Iberian Pyrite Belt (IPB). Consequently, the sediments at the Alqueva reservoir (SE Portugal) in the Guadiana Basin are potentially contaminated by trace elements, which make important: (i) to characterize the status of trace element pollution of the sediments; (ii) to evaluate the mobility and the bioavailability of As, Cd, Cu, Cr, Pb and Zn; and (iii) to assess the environmental risk associated with the total and bioavailable concentrations of trace elements, using the sediment quality guidelines (SQGs) and the risk assessment code (RAC). Metal enrichment factors (EF) and geoaccumulation indexes (I geo ), determined taking into account the regional background levels, revealed that, among the metals analyzed, Cd contributed the highest to pollution levels followed by Pb and As. Despite the trace element contamination of the Alqueva sediments, the sequential extraction showed that most of them are found in the oxidizable and residual fractions, which indicates that they are sparingly bioavailable, with exception of Cd (acid-labile fraction) and Pb (reducible fraction). Based on the RAC, Cd was the only metal that presented a high risk, while Pb, As and Zn showed a medium risk. Moreover, the SQGs revealed the existence of certain areas of extremely high risk, particularly related to high concentrations of total As and, in less extent, of Pb and Cd, associated with AMD, wastewater discharges and runoff of plant protection products from agricultural fields located near the reservoir.