Fluorescence spectroscopy to evaluate the removal of dissolved organic matter (DOM) via biofiltration under varying operational and water quality conditions was investigated in this study. Three feedwaters (lake, river, and wastewater effluent) were examined at three temperatures (6, 22, and 28 °C) and three empty bed contact times (EBCTs; 5, 15, and 30 min) to simulate seasonal and operational variations experienced by utilities. Parallel factor (PARAFAC) analysis was used to characterize the fluorescing DOM and to determine the component removal. Three components were found in the lake and river feedwaters, and four components were found in the wastewater effluent. In natural source waters, protein‐like components were removed most effectively via biofiltration. In the wastewater effluent, a humic‐like component common in nutrient‐rich waters was removed most effectively, and the protein‐like component was removed second most effectively. Increased EBCT and temperature increased the removal efficiency of bulk DOM and the protein‐like components.