The rapid increase in the production of electrical and electronic equipment, along with higher consumption of these products, has caused defective and obsolete equipment to accumulate in the environment. In this research, bioleaching of spent lithium-ion batteries (LIBs) used in laptops is carried out under two-step condition based on the bacterial activities of a mixture of Acidithiobacillus ferrooxidans and Acidithiobacillus thiooxidans. First, the best inoculum ratio of two acidophilic bacteria for the mixed culture is obtained. Next, adaptation is carried out successfully and the solid-to-liquid ratio reaches 40 g L−1. Response surface methodology is utilized to optimize the effective variables of initial pH, iron sulfate and sulfur concentrations. The maximum recovery of metal is about 99.2% for Li, 50.4% for Co and 89.4% for Ni under optimum conditions of 36.7 g L−1 iron sulfate concentration, 5.0 g L−1 sulfur concentration and initial pH of 1.5 for the best inoculum ratio of 3/2. Results of FE-SEM, XRD and FTIR analysis before and after bioleaching confirm that bacterial activity is a promising and effective route for metal recovery from spent LIBs. Toxicity assessment tests demonstrate the suitability of the bioleached residual as a nonhazardous material that meets environmental limitations for safe disposal.