Recent investigations indicate the relevance of extracellular polymeric substances (EPS) in terms of fouling of low-pressure membranes in advanced wastewater treatment. In this study, the high impact of the macromolecular fraction of effluent organic matter on fouling was confirmed in cross-flow ultrafiltration experiments using secondary effluent with and without autochthonous biopolymers. A method for the extraction of a natural mixture of EPS derived from the bacterium Sinorhizobium sp. is presented. Ultrafiltration of solutions of this bacterial EPS extract revealed a correlation between the concentration of EPS and the loss of permeate flux. However, in ultrafiltration tests using extracted bacterial EPS in a model solution as well as in secondary effluent without autochthonous biopolymers, the extent of membrane fouling was not identical with the fouling provoked by secondary effluent organic matter, although the biopolymer concentrations were comparable. The differences in the fouling behaviour of the extracted bacterial EPS and effluent organic matter are considered to be due to different compositions of the biopolymer fraction in terms of proteins, polysaccharides, and other organic colloids, indicating a particular impact of proteins on ultrafiltration membrane fouling.