Manganese removal in drinking water biofilters is facilitated by biological and physico-chemical processes, but knowledge regarding the relative role of these mechanisms during start-up is very limited. The aim of this study was to identify the dominant process for manganese removal occurring during the start-up period of sand filters with and without inoculation by addition of matured sand collected from an operating groundwater-based waterworks. Inoculation with matured filter sand is frequently used to accelerate the start-up in virgin biofilters and to rapidly obtain compliant water quality. The non-inoculated filter took 41 days to comply with manganese quality criteria, whereas the inoculated filter with 20% matured sand showed removal from Day 1 and compliance from Day 25. By Day 48, the inoculated filter showed two times higher manganese removal rates and manganese oxides deposits. Using sodium azide as an inhibitor of microbial activity, it was found that manganese removal in the non-inoculated filter was dominated by biological processes, whereas physico-chemical processes were of more importance in the inoculated filter (Day 35, 39 and 48). 16S rDNA sequencing of the microbiota collected during filter maturation indicated a limited immediate effect of inoculation on the microbial community developed on the remaining filter material.