<p>Iron-sulfur cycle bacteria are considered the principal participants in the regulation of iron and sulfur cycles, ubiquitously found in diverse natural ecosystems. This study concentrated on the spatial distribution patterns of iron-sulfur bacteria in the acid mine drainage (AMD) sediments, compared with AMD-impacted river sediments, and evaluated the potential influences of iron-sulfur bacteria on the metals in the Shandi River basin. The results showed that the water and sediments near the mine from the Shandi River basin had been seriously polluted by heavy metals and sulfate. Specifically, the Nemerow index (P) exceeded 5, and the comprehensive potential ecological risk factor (RI) surpassed 600. The sediment samples collected exhibited a profusion of iron-sulfur cycle bacteria, with the abundance of these organisms being higher within river sediments compared to AMD sediments, particularly for iron-sulfur reducing bacteria. The results of correlation and redundancy analysis showed that most metals had an impact on the abundance of iron-sulfur cycle microorganisms in different degrees. Meanwhile, SEM–EDS analysis revealed the presence of sulfate minerals in diverse forms in sediments, which might be biogenic. All of findings indicated that iron-sulfur cycle bacteria might regulate the forms of metal and sulphur, fixed most metals and sulfate, and further influencing the synthesis and phase transition of sulfate minerals in the sediments. This study confirmed the ecological values of iron-sulfur bacteria, which will be help for bioremediation of AMD contaminants in Shandi River basin.</p>