Abstract: The effect of biofilms on pH and phosphorous in the water-sediment interface were studied for sediments and benthic biological samples from the city sewerage system. The pH values were measured in situ on the interface by the pH microprofile method with an Ir/IrO₂ microelectrode. The biological utilized P content from upper and middle extracting solutions of sediments was determined by Inductively Coupled Plasma-Atomic Emission Spectrometry (ICP-AES). In the experiment, the biofilms primarily consisted of genera Oscillatoria Nitzschia and bacteria Biofilms, which caused an increase of the pH and concentration of phosphorous and the variation of pH and phosphorous concentration in the water across the sediment-water interface. Phosphorous was concentrated in the pore water of the biofilm. Compared to the conditions without biofilms, biofilms provided an opposite coupling relation between the pH and phosphorous concentration in pore water. Without the biofilms, the release of phosphorous from sediments and phosphorous content in the pore water increased with the pH changing from neutral to acidic. Conversely, the biofilm process enhanced the release of phosphorous from sediments and phosphorous content in the pore water with the pH changing from acidic to neutral. Based on the relationship between the pH and phosphorous concentrations, the role of biofilms can be divided into two distinct areas: biological fixation area and biological scavenging area in surface sediments.