Understanding the fate of effluent organic matter (EfOM) and natural organic matter (NOM) through riverbank filtration is essential to assess the impact of wastewater effluent on the post treatment requirements of riverbank filtrates. Furthermore, their fate during drinking water treatment can significantly determine the process design. The objective of this study was to characterise bulk organic matter which consists of EfOM and NOM during riverbank filtration using a suite of innovative analytical tools. Wastewater effluent-derived surface water and surface water were used as source waters in experiments with soil columns. Results showed the preferential removal of non-humic substances (i.e. biopolymers) from wastewater effluent-derived surface water. The bulk organic matter characteristics of wastewater effluent-derived surface water and surface water were similar after 5 m soil passage in laboratory column experiment. Humic-like organic matter in surface water and wastewater effluent-derived surface water persisted through the soil passage. More than 50% of total dissolved organic carbon (DOC) removal with significant reduction of dissolved oxygen (DO) was observed in the top 50 cm of the soil columns for both surface water and wastewater effluent-derived surface water. This was due to biodegradation by soil biomass which was determined by adenosine triphosphate (ATP) concentrations and heterotrophic plate counts. High concentrations of ATP in the first few centimeters of infiltration surface reflect the highest microbial activity which correlates with the extent of DOC reduction. Good correlation of DOC removal with DO and biomass development was observed in the soil columns.