In the present work, the effects of different energy substrates and nickel ions (Ni2+) and cadmium ions (Cd2+) on the growth of Acidithiobacillus ferrooxidans (A. ferrooxidans) were investigated. Ferrous sulphate (FeSO4) was the optimum energy substrate for A. ferrooxidans growth, among the selected substrates. When cultured together with FeSO4 and sulphur (S), A. ferrooxidans first oxidised the ferrous ions (Fe2+), and the S was utilised as the concentration of Fe2+ decreased. After adapting to culture with Ni2+ and Cd2+, A. ferrooxidans presented good tolerance to both ions, with the maximum concentration reaching 4.11 g/L Ni2+ and 1.69 g/L Cd2+. A preliminary simulation of industrial application was also performed on used Ni-Cd batteries. With bioleaching, the highest concentrations of Cd2+ and Ni2+ were 3003 mg/L at day 8 and 1863 mg/L at day 14, respectively.