Sulfate-reducing bacteria (SRB) and iron minerals are widespread in subsurface environments, where their mediated Fe and S transformations are crucial for contaminant immobilization. However, the mechanism mediated by SRB to transform natural iron minerals into reduced iron–sulfur compounds and the contaminant removal capacity of the transformation products remain unclear. Herein, the mechanism of native SRB-mediated transformation of iron-minerals from natural aquifer media into biogenic ferrous sulfide (FeS) was revealed and the Cr(VI) removal performance of the transformation product was evaluated. The results showed that Fe2+ production, sulfate reduction, and sulfide production occurred sequentially (rather than simultaneously) in the liquid phase. This suggests new processes and mechanisms for iron-mineral transformation: first, iron minerals dissolve to produce Fe2+ under enzymatic action rather than sulfide reduction; then, the generated Fe2+ accelerates sulfate reduction by SRB, producing sulfide in large amounts; finally, sulfide combines rapidly with Fe2+ to produce FeS. Elevated temperatures markedly shortened the required transformation time to start: maximum Fe production occurred at 18–24 days, 6–12 days, and 0–6 days at 10 °C, 20 °C, and 35 °C, respectively. Ambient temperature strongly affected SRB community composition, with Desulfosporosinus dominant at 10 °C and 20 °C and Desulfotomaculales prevalent at 35 °C. Sequential extraction, scanning electron microscopy, X-ray photoelectron spectroscopy, and X-ray diffraction analyses confirmed FeS as the main transformation product, which exhibited a highly efficient Cr(VI) removal capacity. Extending transformation time or increasing transformation temperature enhanced the Cr(VI) removal capacity of the transformation product. In column experiments, in-situ stimulation of SRB growth in aquifer media is able to form FeS, the Cr(VI) removal capacity of FeS reached up to 90.1 mg(Cr(VI))/kg(media). These findings suggest that biostimulation of SRB to mediate the in-situ transformation of iron-minerals to FeS has great potential for remediation of contaminated groundwater.