This study is a first attempt from a toxicological perspective to put forward, in general terms and through explanations, two-metal interactions of Cd(II), Cu(II), and Zn(II) with indigenous Acidithiobacillus thiomidans. According to the values of the critical concentration (CC), EC50, and slope factor B obtained in dose-response analysis, synergistic interactions rather than antagonism were found to have evolved for all the metallic cations as the combined effect of binary metal toxicity to indigenous A. thiooxidans. As the fixed-level factor metal increased, almost all the dose-response curves tended to shift toward the left (i.e. the highly toxic side). Compared with the single metal-bearing cultures, significant observed decreases in the critical concentration, resulting in a 100% toxic response for the binary metal systems, also strongly indicated synergistic interactions. In comparison, divalent zinc ion inevitably acted less synergistically than copper and cadmium ion. The toxicity ranking of three individual divalent cations in the binary metal systems with respect to indigenous A. thiooxidans is Cu>Cd>>Zn. This assessment clearly indicates viable operation criteria for metal bioleaching for mine wastewater treatment, suggesting a feasible strategy for biotreatment using acidophilic Thiobacilli A. thiooxidans.