The use of biofilters to produce drinking water from anaerobic groundwater is widespread in some European countries. A major disadvantage of biofilters is the long start-up period required for virgin filter medium to become fully functional. Although individual aspects of biofilter start-up have previously been investigated, no comprehensive study in full scale using inherent inoculation has previously been documented. A thorough investigation of a full-scale drinking water biofilter was carried out over 10 weeks of start-up. The many spatial and temporal changes taking place during start-up were documented using a holistic approach. In addition to collection of many samples over time (frequency) and space (filter depth), this study entailed the use of multiple sample media (water, backwash water and filter media) and multiple types of analyses (physical, chemical and microbiological). The decrease in filter effluent concentrations of individual substances to compliance levels followed a specific order that was shown to coincide with the spatiotemporal development of bacteria on the filter media. Due to the abiotic nature of the iron removal process, iron disappears at the earliest in the start-up period followed by substances that require growth of microorganisms. Ammonium disappears next, with nitrite appearing briefly near the end of ammonium removal, followed by manganese. The thorough overall picture obtained by these efforts provides guidance for optimization and monitoring of the start-up. Guidance for optimization includes shortening the start-up by focusing on kick-starting the ammonium removal; limiting the monitoring burden (at-line measurements of ammonium in finished water supplemented with manual manganese measurements when ammonium removal is complete); and improving filter design by isolating the removal processes in separate, smaller filters.