Coal mine drainage (CMD) is a serious threat to the environment of Appalachia in the United States, releasing large volumes of acid and metal contaminants (e.g., Mn, Fe). Passive limestone-based treatment systems are the most cost-effective method for manganese(II) (Mn(II)) removal from CMD. The success of passive Mn(II)-removal systems has been variable due to a poor understanding of the mechanisms of Mn removal and the microbial communities involved. We selected one Mn-removal system from northwestern Pennsylvania that treats an exceptionally high concentration of 150 mg/L Mn(II), and conducted laboratory experiments to evaluate the relative importance of abiotic versus biotic processes responsible for Mn removal, and to evaluate the relative importance of bacteria versus fungi on biological Mn(II) oxidation. We found that while abiotic processes such as Mn(II) sorption and heterogeneous oxidation contribute to Mn removal, biological Mn(II) oxidation is the most important process to ensure effective, long-term Mn removal. We also found, that fungal activity accounted for over 80% of Mn(II) oxidation in this Mn-removal bed. We also selected four additional Mn-removal systems from western Pennsylvania for an extensive culture enrichment survey of bacteria and fungi. From this survey, we found that Mn(II)-oxidizing fungi were isolated more readily than Mn(II)-oxidizing bacteria - fungal isolates outnumbered bacterial isolates 84:10 in 3 of the 4 systems, and that fungi were extremely tolerant to elevated concentrations of Mn(II).