Human activities have led to increased levels of various pollutants including metals in aquatic ecosystems. Increase of metallic concentrations in aquatic environments represents a potential risk to exposed organisms, including fish. The aim of this study was to characterize the environmental risk to fish health linked to a polymetallic contamination from former uranium mines in France. This contamination is characterized by metals naturally present in the areas (manganese and iron), uranium, and metals (aluminum and barium) added to precipitate uranium and its decay products. Effects from mine releases in two contaminated ponds (Pontabrier for Haute-Vienne Department and Saint-Pierre for Cantal Department) were compared to those assessed at four other ponds outside the influence of mine tailings (two reference ponds/department). In this way, 360 adult three-spined sticklebacks (Gasterosteus aculeatus) were caged for 28 days in these six ponds before biomarker analyses (immune system, antioxidant system, biometry, histology, DNA integrity, etc.). Ponds receiving uranium mine tailings presented higher concentrations of uranium, manganese and aluminum, especially for the Haute-Vienne Department. This uranium contamination could explain the higher bioaccumulation of this metal in fish caged in Pontabrier and Saint-Pierre Ponds. In the same way, many fish biomarkers (antioxidant and immune systems, acetylcholinesterase activity and biometric parameters) were impacted by this environmental exposure to mine tailings. This study shows the interest of caging and the use of a multi-biomarker approach in the study of a complex metallic contamination.