During wastewater transportation in sewers conversion of organic matter into biomass takes place in bulk water and in bacterial biofilms. The biomass amount, the composition and the properties influences wastewater composition and the subsequent fate in the wastewater treatment plant. Because the biomass consists of both cell biomass and extracellular polymers having different properties, the biomass composition in biofilms from three different gravity sewers is reported here. Cell biomass was only a minor fraction of the organic matter in the biofilms and 70–98% of total organic carbon was found to be extracellular. The macromolecular composition of the biofilm was determined and the major part was protein. Also in the extracellular fraction protein was the largest fraction. Moreover, humic substances, polysaccharide, uronic acids and DNA could be extracted from all biofilm samples into an extracellular fraction. Between 30 and 40% of the COD from the total biofilm sample were not analysed by the methods used. Some variation in the content and composition of extracellular material was found among the different sewer lines. The results demonstrate that biofilm material from sewer lines entering a wastewater treatment plant mainly consists of heterogeneous extracellular organic material with protein as the dominating fraction.