Slow sand filtration (SSF) is one of the oldest biofiltration methods for reducing pathogens and organic matter (OM) in water. Due to its efficiency, affordability, and operational simplicity, SSF remains a widely used approach for producing biologically stable drinking water. Although biological activity plays a role in the removal of OM during SSF, its contribution is poorly constrained. Here, we explored the utility of stable isotopes for investigating this role quantitatively on the scale of an operational filter. First, by combining measurements of concentrations and natural isotopic composition in relevant carbon pools (dissolved and solid, organic and inorganic), we found evidence for OM removal through both retention and subsequent mineralization. However, their relative contributions could not be constrained due to insufficient precision and continuity of available data and incomplete knowledge about the relevant isotope fractionation factors. In the other approach, we therefore used laboratory incubations of SSF cores with