Manganese removal is a typical concern in drinking water production. Biofiltration may be used when treating groundwater sources but the onset of manganese removal in virgin biofilters can vary considerably. The aim of this study was to investigate the effect of different filter media on manganese oxidation and bacterial diversity in biofilters during the start-up. The onset of manganese oxidation in four virgin granular filter media (quartz, calcium carbonate, polystyrene, and manganese oxide) and one matured medium (quartz) was followed during the start-up. Immediate manganese removal was achieved by manganese oxide, while 48, 57 and 72 days were required by virgin quartz, calcium carbonate and polystyrene, respectively. The bacterial community was investigated using DAPI staining, quantitative polymerase chain reaction (qPCR), 16S rRNA gene pyrosequencing, and bacterial enrichments. Bacterial abundance was greatest on polystyrene and matured quartz. Molecular community analysis and bacterial enrichments suggested the presence of manganese oxidizing bacteria on all media coatings after the start-up period. Virgin quartz and calcium carbonate showed similar bacterial communities whereas manganese oxide and polystyrene were distinct. This investigation suggests that when inoculating different filter media with an identical water source, the bacterial diversity and onset of manganese oxidation during start-up is strongly influenced by the filter media type.