Indigenous acidophilic bacteria separated from mine-waste can be used in return for the addition of the reagents like sulfuric acid. Among the tailings bacteria, Acidithiobacillus ferrooxidans and Acidithiobacillus thiooxidans are of the most-studied ones for the bioleaching and bioremediation of elements. In this work, the isolation and characterization of the mentioned bacteria are studied by a proposed biochemical protocol. The sequential cultivation of the soil bacteria in a series of liquid media and solid culture medium cause the separation of bacteria. A biochemical method is used to characterize the isolated strains of the native bacteria. The changes in the Eh, pH, and culture medium color are checked in order to control the bacterial growth in a 9K liquid medium. At the first step of the sequential cultivation, the amount of nutrient broth is the main factor that affects the complete isolation of Acidithiobacillus bacteria. The trivial dosage of nutrient broth does not lead to the desired proliferation of the bacteria in the tailings soil. 8 g L-1 of the nutrient broth is suitable to increase the initial number of bacteria in the soil significantly. In the next steps, the bacteria are separated from the soil, and pure strains of A. ferrooxidans and A. thiooxidans are isolated using a 9K medium. Final pure strains are achieved during the two steps of streak cultivation of bacteria in the soil medium of nutrient agar.