<p>Acid mine drainage (AMD) is recognized as a major environmental and human health hazard. Understanding the responses of Fe/S-metabolizing microorganisms to AMD exposure could elucidate their survival strategies and inform remediation approaches. A terrace exhibiting sharp geochemical gradients induced by AMD was selected to investigate microbial Fe/S-metabolizing responses to AMD-related environmental parameter shifts. Our results demonstrated the AMD tributary exhibited higher pollutant attenuation efficiency (per unit distance) than the AMD-contaminated Shandi River, whereas sediment pollutant levels remained stable. The AMD-contaminated Shandi River showed higher α-diversity indices (OTUs: 4731 vs. 2724; Chao1: 4739 vs. 2745; Shannon: 10.35 vs. 8.82) than the AMD tributary. Microbial community structure diverged significantly across pollution gradients, especially at phylum and genus levels. The abundance of Fe/S-metabolizing microorganisms increased with distance from the pollution source, primarily driven by Fe²⁺, DO, COD, and pH. Furthermore, by integrating the spatial succession patterns of Fe/S-metabolizing taxa with contaminant attenuation dynamics, we identified the R2 section as the optimal zone for implementing sulfate-reducing bacteria (SRB)-based in situ bioremediation along the Shandi River. These findings highlight the profound influence of AMD pollution gradients on sediment microbial communities, clarifying evolutionary mechanisms of Fe/S-metabolizing microorganisms under varying contamination levels. These results provide mechanistic insights into AMD-driven impacts on Fe/S-metabolizing microbial communities and lay a foundation for in situ bioremediation strategies.</p>