The dispersed pollution caused by microplastics (MPs) represents a current and global concern. While the fragmentation of plastic debris into smaller particles occurs in rivers, little MP research is done on freshwater species and is published compared to the marine environment. The Loire River is the longest river in France and is subject to moderate to high anthropic pressure while it represents major societal and economic issues. However, there are not many studies that have been put forward with regards to the effect of environmental MPs (EMPs) on aquatic organisms and no policies have been enacted to monitor the plastic pollution. In this study, freshwater bivalves, Corbicula fluminea, were exposed for 21 days to environmentally relevant concentrations of a mixture of <200 µm MPs generated from plastic litter collected directly along the banks of the Loire River. This mixture was composed of 40% polyethylene (PE), 40% polypropylene (PP), 10% polyethylene terephthalate (PET) and 10% polyvinylchloride (PVC) (mass percentage). Ecotoxicological effects were assessed from the individual to the molecular levels on several endpoints: condition index, filtration efficiency, enzyme activities, lipid peroxidation, energy reserves and gene expression. The ingestion of EMPs caused damages at the biochemical level. Indeed, we reported an increase in catalase activity in gills and digestive mass, a decrease in TBARs in gills, a decrease in acetylcholinesterase activity in the digestive mass, a decrease of glycogen and lipid contents in the whole organisms and a significant induction of the expression of gst, cat, mp, acp genes. The current results suggest therefore that long-term exposure to realistic doses of EMPs causes toxicity towards freshwater benthic biota. The analysis of biomarker activities and the analysis of gene expression are complementary to prevent the effects of a plastic contamination at higher biological levels in aquatic organisms.