The main aim of this work was to stimulate biofilm formation in the presence of wastewater containing aromatic compounds with different toxicities (EC50). The results indicated that wastewater with an EC50 value > 85% accelerates the attachment of bacteria onto the bio-carriers because the toxic wastewater stimulates the production of extracellular polymeric substances (EPS) from the seed sludge. In order to understand the role of EPS on biofilm development, experiments were conducted using the seed sludge, from which the soluble, loosely bound, and tightly bound EPS were removed. The soluble EPS fraction was determined to be crucial for biofilm development. Firmicutes bacterium and Clostridium chromoreductans survived and were enriched in the formed biofilms in our study, which can resist toxic aromatics.