The effect of anionic surfactants on organic matter degradation during enhanced in-sewer purification by porous media was evaluated in this study. Surfactants, wherein the anionic subgroup comprises a significant portion of surfactant load in domestic wastewater, are anticipated to affect biofilm activity during enhanced in-sewer purification negatively. To evaluate this, synthetic feed containing a fixed concentration of readily biodegradable organic matter and different anionic surfactant concentrations was supplied intermittently to an airtight channel intended for enhanced in-sewer purification for 32 days. Dodecylbenzene sulfonate, as sodium dodecylbenzene sulfonate, was used as a model anionic surfactant. Results showed that increased anionic surfactant concentration up to 160 mg L −1 decreased aerobic organic matter degradation rate by up to 36% of the initial rate. It was inferred from the compositional analysis of effluent surfactants by liquid chromatography tandem mass spectrometry that the accumulation of possible inhibitory intermediate products may have caused decline in rates over time. Recovery of aerobic activity related to organic matter degradation was observed with continued operation under decreasing influent anionic surfactant concentration. Findings highlight that although increased surfactants concentrations negatively affected aerobic activity, aerobic organic matter was still continuously degraded aerobically.