Experimental investigations were performed to determine the possibility of simultaneous biological nitrogen and phosphorus removal during various biofilm processes in conjunction with biofilm characterisation, especially extracellular polymeric substance (EPS). Since biologial nitrogen removal requires an alternating exposure of anaerobic-anoxic-oxic conditions in the bulk liquid that surrounds the biofilm growth media, a sequencing batch reactor (SBR)-type operation was used. Various materials including expanded clay, polystyrene, polyurethane, and acrylic materials were used as the biofilm growth support medium. Simultaneous nitrogen and phosphorus removal was possible with SBR, but it was postulated that nutrient removal efficiencies varied with film thickness. Thinner biofilm promoted nitrification and phosphorus removal, but thicker biofilm enhanced denitrification and reduced phosphorus removal. EPS contents were similar regardless of support media types or biofilm configuration, but EPS contents gradually increased as the film growth continued after backwashing. EPS contents were increased with increased nitrogen removal, but it was difficult to define its relation with phosphorus removal. In addition, suspended solids removal was correlated well with the EPS content in the biofilms.