Slow Sand Filtration is popular in drinking water treatment for the removal of a wide range of contaminants (e.g., particles, organic matter, and microorganisms). The Schmutzdecke in slow sand filters (SSFs) is known to be essential for pathogen removal, however, this layer is also responsible for increased head loss. Since the role of deeper layers in bacteria and virus removal is poorly understood, this research investigated the removal of E.coli WR1 and PhiX 174 at different depths of a full-scale SSF. Filter material from top (0–5 cm), middle (5–20 cm) and deep (20–35 cm) layers of an established filter was used in an innovative experimental set-up to differentiate physical-chemical and biological removal processes. In the analysis, we distinguished between removal by biological activity, biofilm and just sand. In addition, we modelled processes by a one-side kinetic model. The different layers contributed substantially to overall log removal of E.coli WR1 (1.4–1.7 log10) and PhiX 174 (0.4–0.6 log10). For E.coli WR1, biological activity caused major removal, followed by removal within biofilm and sand, whereas, removal of PhiX 174 mainly occurred within sand, followed by biofilm and biological activity. Narrow pore radii in the top layer obtained by micro-computed tomography scanner suggested enhanced retention of bacteria due to constrained transport. The retention rates of E.coli WR1 and PhiX 174 in top layer were four and five times higher than deeper layers, respectively (kret 1.09 min−1 vs 0.26 min−1 for E.coli WR1 and kret 0.32 min−1 vs of 0.06 min−1 for PhiX 174). While this higher rate was restricted to the Schmutzdecke alone (top 5 cm), the deeper layers extend to around 1 m in full-scale filters. Therefore, the contribution of deeper layers of established SSFs to the overall log removal of bacteria and viruses is much more substantial than the Schmutzdecke.