The acidophilic sulfur oxidizing bacterium (SOB), Acidithiobacillus ferrooxidans, has been found to stimulate elemental sulfur (S⁰) oxidation and mineral weathering in alkaline Fe ore tailings. However, A. ferrooxidans growth and activities depend on the pH conditions surrounding their interfaces with minerals. The present study aimed to investigate how pH influences bacterial growth and functions in Fe ore tailings. A simulated aquatic ‘homogeneous’ incubation system was initially adjusted into acidic (pH 4), neutral (pH 7) and alkaline (pH 9) conditions, which mimicked the microenvironmental conditions of the water-cell-mineral interfaces in the tailings. It was found that A. ferrooxidans grew well and oxidised S⁰ under the prevailing and initially acidic conditions (pH < 6). These stimulated the weathering of biotite and amphibole-like minerals and the formation of nanosized jarosite and ferrihydrite-like minerals mediated by extracellular polymer substrate (EPS). In contrast, the initially neutral/alkaline pH conditions (i.e., pH > 7) with the presence of the alkaline tailings restricted SOB growth and functions in S⁰-oxidation and mineral weathering. These findings suggest that it is essential to prime acidic conditions in microenvironments to support SOB growth, activities, and functions toward mineral weathering in tailings, providing critical basis for enrolling SOB in eco-engineered pedogenesis in tailings.