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This study aims to use geochemical, mineralogical, ecotoxicological and biological indicators for a comprehensive assessment of the ecological risks related to the mobility, ecotoxicity and bioavailability of potentially harmful elements in the Lousal mining district. Particularly, toxicity was evaluated using four assays: algae, cytotoxicity assays with HaCaT cell line (dermal), earthworms and Daphnia magna. The geochemical and mineralogical characterization of the studied area shows that the mine wastes underwent intense weathering processes, producing important contamination of the adjacent soils, which also led to the release and mobilization of PHEs into nearby water courses. Total PTE results indicate that the soils affected by mining activities were highly contaminated with As and Cu, while Zn and Pb content ranged from low to very high, depending on the analyzed samples. Cadmium levels were found to be very low in most of the soil samples. The test using Daphnia magna was the most sensitive bioassay, while the Eisenia foetida test was the least sensitive. Except for the LOS07 soil sample, the rest of the soils were classified as “High acute toxicity” and “Very high acute toxicity” for aquatic systems. The results in HACaT cells showed results similar to the ecotoxicological bioassays. The application of biotests, together with geochemical and mineralogical characterization, is a very useful tool to establish the degree of contamination and the environmental risk of potentially harmful elements.