Knowledge on the impact of plastics on aquatic organisms are generally based on the effect evaluation induced by standard materials. This reductionist approach ignores the different characteristics of plastics when in the ecosystems, as well as the possible effect of chemicals adsorbed on their surface. In this study, we carried out the quali-quantitative evaluation of 5 plastic mixtures collected along the Lambro River (Northern Italy), that crosses one of the higher anthropized and industrialized Italian areas, contemporarily evaluating their environmental hazard by an ecotoxicological approach. We collected plastics at 5 sampling points along the river using two twin plankton nets with a mesh of 300 µm, repeating the sampling for 3 days in a week. Plastics collected from one net were quantified and characterized through the Fourier Transform Infrared Microscope System (µFT-IR), while plastics from the second one were separated from interfering materials and subsequently used for in vivo exposures. We exposed the freshwater mussel Dreissena polymorpha to these plastic mixtures for 21 days, evaluating the toxicity at the end of exposures using an integrated approach based on the measure of some endpoints from the cellular and molecular levels to the whole organism. The obtained results showed an increasing trend of plastic concentration along the Lambro course, with an estimate of about 4,700,000 plastics released daily into the Po River, the main Italian watercourse. After the exposure to “environmental” plastic mixtures we observed a great mortality coupled with significant decrease in cell viability in mussels exposed to some mixtures, as well as an imbalance of the oxidative status, with a significant increase of both catalase activity and protein carbonylation and a modulation of proteins mainly involved in the oxidative stress response. This work aimed to propose a new tool to address the Environmental Risk Assessment for these emerging pollutants.