The effects of Fe(III) on the biofilm mass and activity, the biofilm micromorphology as well as the composition and functional groups characteristics of extracellular polymeric substances (EPS) in biofilm were investigated in laboratory-scale fixed bed biofilm reactors. The results showed that 2 mg/L of Fe(III) promoted the biofilm mass and improved the biofilm activity, but 16 mg/L of Fe(III) adversely affected biofilm development. Scanning electron microscopy (SEM) study indicated a high concentration (16 mg/L) of Fe(III) led to signiﬁcant reduction of the ﬁlaments, great promotion of the EPS secretion in biofilm. The result of the EPS composition suggested 2 mg/L of Fe(III) increased soluble EPS and loosely bound EPS which contributed to the microbial aggregation, while 16 mg/L of Fe(III) promoted tightly bound EPS production unfavourable for substrate mass transfer. Fourier transform infrared spectroscopy and X-ray photoelectron spectroscopy analysis demonstrated that Fe(III) exerted a significant influence on the –CONH– groups of proteins and the C–O groups of polysaccharides in EPS. This study reveals that Fe(III) influences biofilm development and activity not only by directly impacting the microbial physiology but by indirectly affecting the EPS constituents, and it helps to provide theoretical guidance for iron ion containing wastewater treatment.