Iron (Fe) and manganese (Mn) are common elements of concern in groundwater in the Canadian Prairies. Biological filtration that stimulates indigenous Fe- and Mn-oxidizing microorganisms that are naturally present in groundwater is often considered a cost-effective water treatment option. One of the challenging aspects of biological treatment is that low temperatures significantly hinder microbial metabolic activity. This study focuses on enhancing cold-adapted, indigenous microbial populations for Fe and Mn oxidation at the in situ low temperatures (8 °C) of a pilot-scale biofilter at the Langham water treatment plant in Saskatoon. The pilot-scale biofiltration system consists of two aerated biofilters connected in series, designed to remove Fe in Filter 1 and Mn in Filter 2. The growth of biofilms was promoted either on conventional plastic filter media or on anthracite. Rapid oxidization of iron occurred through both filters in one month (99% removal, p < 0.05). After several months, Mn removal was successfully achieved in Filter 2 when it contained anthracite (97% removal, p < 0.05). Scanning Electron Microscopy/Energy Dispersive X-ray Spectroscopy (SEM/EDS) and Inductively Coupled Plasma Mass Spectrometry (ICP-MS) analyses confirmed the removal of Fe and Mn to meet water quality criteria. Adsorption of Mn on anthracite, which was confirmed by an additional batch experiment, likely promoted the biological removal, bacterial immobilization, and/or physicochemical removal of Mn in Filter 2. Culture-dependent microbial assessments coupled with the leucoberbelin blue method indicated the presence of Mn-oxidizing bacteria in the biofiltration system.