Acid mine drainage (AMD) generates acidic wastewater containing high concentrations of sulfate and dissolved metals, which can cause severe environmental degradation. Sulfate-reducing bacteria (SRB) can be applied in biological sulfate reduction for wastewater treatment because they neutralize acidity and transform dissolved metals into sulfide precipitates. This study evaluated SRB performance under two acclimatization strategies and varying Fe and Mn concentrations in different reactor operation modes. Gradual acclimatization was conducted by stepwise sulfate elevation to approximately 2,000 mg/L, whereas shock acclimatization involved direct exposure to 2,000 mg/L sulfate. Batch reactors were operated for 14 days with Fe and Mn concentrations ranging from 10 to 100 mg/L, followed by continuous reactor operation for 22 days to assess medium-term performance. Gradual acclimatization resulted in higher sulfate reduction, greater pH increase, and better sediment stability than shock acclimatization. In batch operation, the highest sulfate reduction efficiency was 46.58% with a reduction rate of 29.13 mg/L·day under Fe-dominant conditions, namely 10 mg/L Fe without Mn. By contrast, Mn dominance reduced the efficiency to 40.62%. Higher Fe and Mn concentrations, including the inhibitory concentration (IC₅₀) and maximum tolerable concentration (MTC), further suppressed performance, whereas extreme concentrations of 100 mg/L resulted in sulfate removal mainly through chemical precipitation. Continuous reactors demonstrated greater resilience to metal stress. Overall, gradual acclimatization and continuous operation improved AMD treatment performance, with Mn identified as the more toxic inhibitor of SRB activity.