The addition of biodegradable carbon sources to sand filters can enhance microbial activity but may lead to substrate clogging, a major operational problem. In laboratory-scale soil columns emulating vertical up-flow filters, the clogging effect of two readily biodegradable organic substrates—sucrose as a sugar source and ethanol as an alcohol source—were examined with coarse sand as the substrate medium. Wastewater without the addition of supplemental organics and a ‘control’ treated with tap water were monitored as references. Changes in saturated hydraulic conductivity were measured for all treatments over time. Other parameters that can influence the clogging rate, including temperature, dissolved oxygen, chemical oxygen demand, protein, and polysaccharides, were measured in the influent and effluent wastewater on a weekly basis. At the end of the clogging experiment, the main layer of each filter bed was separated into three sections and saturated hydraulic conductivity, organic matter content, and protein and polysaccharide concentrations were measured in each section. The rate of clogging development in the columns depended on treatment, with ethanol-treated cores clogging more quickly than sucrose-treated cores. Wastewater-treated cores took far longer to clog and the tap water control did not clog, but the saturated hydraulic conductivity declined by 60% over a year. Saturated hydraulic conductivity within the treated cores declined far less than the calculated decline in saturated hydraulic conductivities for the entire cores at the end of the experiment, indicating that clogging in the vicinity of the inlet plate by microbial mats was a major factor influencing the reduction in flow through the columns. To reduce bio-clogging in inlet filters, it may be advantageous to inject organic amendments directly into the bed, rather than pass them through the filters, as is usually the case.