Slow sand filters (SSFs) are increasingly recognized for enhancing the biological stability of drinking water. While research has historically focused on the top layer (Schmutzdecke) of SSFs, the contribution of deeper filter depths in removing dissolved organic carbon (DOC) and ammonium (NH4+) has recently been acknowledged. This study investigated the occurrence and potential pathways of DOC release in mature full-scale, and young laboratory SSFs. The top layer (5 cm) reduced the easily biodegradable DOC, mainly low-molecular-weight (LMW) acids and building blocks. The middle layers (20–60 cm) released DOC, particularly LMW acids and neutrals, at depths where nitrification was nearly complete. This release occurred in both mature and young SSFs and may result from bacterial activity under carbon or nitrogen limitation or from the transformation of slowly degradable DOC into labile forms. Whatever the precise mechanism of release, the bottom layers (60–90 cm) subsequently removed this released DOC and reduced PO43– to ultralow levels, highlighting the importance of the deepest layers in maintaining effluent quality. This study provides the first evidence of biodegradable DOC release in SSFs and emphasizes the need to better understand its implications for carbon cycling and removal processes in biological filters.