Dissolved organic matter (DOM) is a complex mixture of carbon-based compounds present in natural aquatic systems, which significantly affects drinking water treatment processes. Biofiltration, utilising biologically active beds of porous medium, offers a low-energy and low-chemical solution for controlling bioavailable DOM. However, the impact of microbial community composition on DOM degradation in biofilters remains poorly understood. This study aimed to explore the abilities of microbial communities from the top, middle, and bottom (TOP, MID, and BOT) of a biofilter to process DOM. We showed varying growth rates on the DOM, with bottom community exhibiting the highest cell abundance at the end of the experiment (1.83 × 106 ± 9 × 103; 2.06 × 106 ± 1 × 104; 2.15 × 106 ± 7 × 103 cells/mL for the TOP, MID, and BOT, respectively). The three communities showed different preferences for utilising specific DOM fractions, with the bottom community targeting more complex ones. The microbial communities from the bottom of the biofilter had a higher relative abundance of the Curvibacter genus, suggesting it could play a crucial role in degrading complex DOM fractions. These findings highlight the influence of microbial community composition on DOM degradation in biofilters, providing valuable insights for optimising their performance.