As the number of legal and illegal mining sites increase, integrative methods to evaluate the effects of mining pollution on Andes-Amazonia freshwater ecosystems are paramount. Here, we sampled water and sediments in 11 sites potentially affected by mining activities in the Napo province (Ecuador). The environmental impacts were evaluated using four lines of evidence (LOEs): water physico-chemical parameters; metal exposure concentrations; macroinvertebrate community response (AAMBI); and toxicity by conducting bioassays with Lactuca sativa and Daphnia magna. Overall, dissolved oxygen and total suspended solids were, under (<80%) and above (>130 mg/Ls) quality standards. Ag, Al, As, Cd, Cu, Fe, Mn, Pb and Zn in water and V, B and Cr in sediments were detected above quality standards. Nine out of eleven sites were classified as having bad environmental quality based on the AAMBI. Ranges of L. sativa seed germination in both water (37% to 70%) and sediment (0% to 65%), indicate significant toxicity. In 5 sites, neonates of D. magna showed a 25% reduction in survival compared to the control. Our integrated LOEs index ranked sites regarding their environmental degradation. Given the importance of the Andes-Amazon region, we recommend environmental impact monitoring of the mining expansion using multiple LOEs.