To better understand biofiltration, concentration profiles of various natural organic matter (NOM) components throughout a pilot-scale drinking water biofilter were investigated using liquid chromatography – organic carbon detection (LC-OCD) and fluorescence excitation and emission matrices (FEEM). Over a 2 month period, water samples were collected from six ports at different biofilter media depths. Results showed substantial removal of biopolymers (i.e. high molecular weight (MW) NOM components as characterized by LC-OCD) and FEEM protein-like materials, but low removal of humic substances, building blocks and low MW neutrals and low MW acids. For the first time, relative biodegradability of different NOM components characterized by LC-OCD and FEEM approaches were investigated across the entire MW range and for different fluorophore compositions, in addition to establishing the biodegradation kinetics. The removal kinetics for FEEM protein-like materials were different than for the LC-OCD-based biopolymers, illustrating the complementary nature of the LC-OCD and FEEM approaches. LC-OCD biopolymers (both organic carbon and organic nitrogen) and FEEM protein-like materials were shown to follow either first or second order biodegradation kinetics. Due to the low percent removal and small number of data points, the performance of three kinetic models was not distinguishable for humic substances. Pre-filtration of samples for FEEM analyses affected the removal behaviours and/or kinetics especially of protein-like materials which was attributed to the removal of the colloidal/particulate materials.