Natural biofilm can be a suitable medium for the monitoring of pollutants. Limited information is currently available regarding the occurrence of per- and polyfluoroalkyl substances (PFAS) in periphytic biofilm and low-trophic level organisms of freshwater ecosystems. In this study, surface water, biofilm, phytoplankton, and freshwater snails were collected from Taihu Lake, China, and characterized for 16 PFAS, including legacy compounds (PFSAs/PFCAs) and PFAS of emerging concern (fluorotelomer sulfonates and F-53B). The colonized biofilms effectively bioaccumulated PFAS from water, with the total concentration (∑PFAS) in the range of 1.96-20.1 ng/g wet weight, and the bioaccumulation factor increased with the PFAS log Kow values. As compared with phytoplankton, the ∑PFAS in biofilms displayed a stronger correlation with those in water. PFAS distinctly biomagnified from the biofilm to freshwater snail, with the biomagnification factor in the range of 3.09 ± 2.03 - 17.8 ± 10.2, implying the important role of biofilm in PFAS transfer in aquatic environment. Extracellular proteins production in biofilm increased with the water PFAS concentrations. The total extracellular polymeric substances (EPS) content increased with the water PFAS concentration firstly and then declined to a steady level, while the algal chlorophyll level exhibited a similar relationship with the PFAS in biofilm. High PFAS levels were also associated with depressed alpha diversity of fungal community in biofilms. Biofilm appears as a relevant indicator to characterize the occurrence of PFAS in aquatic ecosystems.