In this study, 50 pesticides were analyzed in the Ebro River basin in 2010 and 2011 to assess their impact in water, sediment and biota. A special emphasis was placed on the potential effects of both, individual pesticides and their mixtures, in three trophic levels (algae, daphnia and fish) using Risk Quotients (RQs) and Toxic Units (TUs) for water and sediments. Chlorpyrifos, diazinon and carbendazim were the most frequent in water (95, 95 and 70% of the samples, respectively). Imazalil (409.73 ng/L) and diuron (150 ng/L) were at the highest concentrations. Sediment and biota were less contaminated. Chlorpyrifos, diazinon and diclofenthion were the most frequent in sediments (82, 45 and 21% of the samples, respectively). The only pesticide detected in biota was chlorpyrifos (up to 840.2 ng g-1). Ecotoxicological risk assessment through RQs showed that organophosphorus and azol presented high risk for algae; organophosphorus, benzimidazoles, carbamates, juvenile hormone mimic and other pesticides for daphnia, and organophosphorus, azol and juvenile hormone mimics for fish. The sum TUsite for water and sediments showed values < 1 for the three bioassays. In both matrices, daphnia and fish were more sensitive to the mixture of pesticide residues present.