The low efficiency and selectivity limitations of biohydrometallurgy technique compel the researchers to explore novel microbial strains acclimated to metal existence site with higher toxicity tolerance and bioleaching capability in order to improve the role of bioleaching process for e-waste management. The current study aimed to explore bioleaching potential of indigenous Bacillus sporothermodurans ISO1; isolated from metal habituated site. The statistical approach was utilized to optimize a variety of culture variables including temperature, pH, glycine concentration and pulp density that impact bio-cyanide production and leaching efficiency. The highest dissolution of Cu and Ag, 78% and 37% respectively, was obtained at 40 °C, pH 8, glycine concentration 5 g L−1, and pulp density 10 g L−1 through One Factor at a Time (OFAT), which was further increased up to 95% Cu and 44% Ag recovery through the interactive effect of key factors in the Response Surface Methodology (RSM) approach. Furthermore, Chemo-biohydrometallurgy approach was utilized to overwhelm the specificity limitation; as higher concentration of Cu in computer printed circuit boards (CPCBs) causes interference to recover other metals. The sequential leaching through ferric chloride (FeCl3), recovered Cu prior to bio-cyanidation by B. sporothermodurans ISO1 and resulted in the improved leaching of Ag (57%), Au (67%), Pt (60%), etc. The current work reports on B. sporothermodurans ISO1, a new Bacillus strain that exhibits highest toxicity tolerance (EC50 = 425 g L−1) than earlier reported stains and has higher leaching potential that can be implemented to large-scale biometallurgical process for e-waste treatment to achieve the agenda of sustainable development goal (SDG) under the strategies of urban mining.