The roles of different extracellular polymeric substance (EPS) fractions in microbial communities, such as periphytic biofilms, are unclear. In this study, three EPS fractions extracted from a periphytic biofilm were employed to investigate their ability to affect Fe2O3 nanoparticle (IONP) toxicity. The addition of IONPs promoted the production of EPS and reactive oxygen species. The bound EPS protected the microbial cells in the periphytic biofilm from oxidative damage caused by IONPs. Different EPS fractions had different roles in affecting IONPs via different mechanisms, specifically, the soluble EPS increased the hydrodynamic diameter of IONPs and promoted IONP aggregation, the flocculation efficiency of the tightly bound EPS increased during exposure to IONPs, and the loosely bound EPS presented high biosorption of IONPs. The binding of IONPs with abundant functional groups was the main combination mode between different EPS fractions and IONPs. The beneficial changes in composition, structure and functional groups of EPS during exposure to IONPs in turn facilitate the elimination of their toxicity. This study contributes to a better understanding of the resistance mechanism of the periphytic biofilm to IONP exposure and the roles of EPS fractions in defending against nanotoxicity.