Increasing metal consumption is driving the introduction of new techniques such as biomining to exploit low grade ores. The biomining impacts notably aquatic ecosystems, yet, the applicability of ecotoxicological tests to study the complex mixture effects of mining waters is insufficiently understood. The aim of the present work was to test if transcriptomic biomarkers are suitable and sensitive for the ecotoxicity assessment of biomining affected waters. The study site had been affected by a multimetal biomine, and the studied water samples formed a concentration gradient of contamination downstream from the biomining site. Cadmium and nickel were used as positive controls in the toxicity tests. Selected transcriptomic biomarkers, previously shown to be differentially regulated by metals, were used to evaluate the ecotoxicity of the water samples. Parallel samples were used to compare the transcriptomic biomarkers with the conventional acute D. magna toxicity test. In the acute test, one sample was acutely toxic to D. magna, when pH was adjusted according to the standard, whereas, in the native pH, three samples caused total immobility. Monooxygenase was up-regulated by the highest concentration of Cd in control samples and three of the water samples. Vtg-SOD was up-regulated by one of the water samples, and catalase by the second highest concentration of Cd. The results show that transcriptomic biomarkers in D. magna can be used as sensitive bioindicators for metal mixture toxicity assessment in complex environmental water samples.