Biological activated carbon (BAC) filters are frequently used in drinking water production for removing dissolved organic carbon (DOC) via adsorption of organic compounds and microbial degradation. However, proper methods are still missing to distinguish the two processes. Here, we introduce reverse stable isotope labelling (RIL) for assessing microbial activity in BAC filters. We incubated BAC samples from three different BAC filters (two granular activated carbon- and one extruded activated carbon-based) in a buffer amended with C-labelled bicarbonate. By monitoring the release of C-CO from the mineralization of DOC, we could demonstrate the successful application of RIL in analysing microbial DOC degradation during drinking water treatment. Changing the water flow rates through BAC filters did not alter the microbial activities, even though apparent DOC removal efficiencies changed accordingly. Microbial DOC degradation activities quickly recovered from backwashing which was applied for removing particulate impurities and preventing clogging. The size distributions of activated carbon particles led to vertical stratification of microbial activities along the filter beds. Our results demonstrate that reverse isotope labelling is well suited to measure microbial DOC degradation on activated carbon particles, which provides a basis for improving operation and design of BAC filters.