Packed bed bioreactors were used to remove soluble manganese from a synthetic mine water as the final stage of an integrated bioremediation process. The synthetic mine water had undergone initial processing using a sulfidogenic bioreactor (pH 4.0–5.5) which removed all transition metals present in elevated concentrations (Cu, Ni, Zn and Co) apart from manganese. The aerobic bioreactors were packed with pebbles collected from a freshwater stream that were coated with black-colored, Mn(IV)-containing biofilms, and their capacity to remove soluble Mn (II) from the synthetic mine water was tested at varying hydraulic retention times (11–45 h) and influent liquor pH values (5.0 or 6.5). Over 99% of manganese was removed from the partly processed mine water when operated at pH 6.5 and a HRT of 45 h. Molecular techniques (clone libraries and T-RFLP analysis) were used to characterize the biofilms and identified two heterotrophic Mn-oxidizing microorganisms: the bacterium Leptothrix discophora and what appears to be a novel fungal species. The latter was isolated and characterized in vitro.