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The Hughes Borehole is located in the bituminous coal region of south-central Pennsylvania and drains an underground mine complex of ca. 2,950 ha. The borehole discharge averages approximately 63 L/s of acidic (pH 4, 230 mg/L acidity) mine drainage that contains elevated concentrations of Fe(II) (100 mg/L). Long-term monitoring of the site showed that biological Fe(II) oxidation occurred without human intervention and, over previous decades, has produced a 0.6 ha iron mound up to 2 m deep. On-mound channel reactors along with laboratory-scale gutter reactors were constructed to determine conditions that can exploit biological Fe(II) oxidation for passive treatment. Dissolved Fe(II) was much more efficiently oxidized from gutter reactors that contained iron mound sediment than ones without any sediment. Close to 100% of the dissolved influent Fe(II) was oxidized in 5–10 h and 75% of the total dissolved Fe was removed. In addition, the reactors performed better as the sediment aged, so that shorter residence times of 1–2 h were also capable of oxidizing substantial amounts of the influent Fe(II). The addition of surface area to the on-mound reactors improved Fe(II) oxidation at residence times of 30 min or less. The results of this study can be used to help design and build treatment systems for low-pH acid mine drainage (AMD) discharges.