Per- and poly-fluoroalkyl substances (PFAS) are an emerging class of persistent organic pollutants that are widespread in aquatic ecosystems and pose a serious threat to aquatic organisms. It is thus crucial to explore the toxicity mechanisms of PFAS to submerged macrophytes and biofilms. In this study, Vallisneria natans (V. natans) was exposed to environmentally relevant concentrations of perfluorooctanoic acid (PFOA) and perfluorooctane sulphonate (PFOS). Results showed that PFAS induced the excessive production of reactive oxygen species, triggering antioxidant responses. V. natans exhibited an improved stress tolerance by altering the biosynthesis of several plant secondary metabolites and the histidine, arginine, proline pathways in response to PFAS exposure. Moreover, PIP1–1, PIP2–2, SLAH1 and SLAH2 genes were upregulated, indicating the activation of aquaporins and slow-type anion channels. The uptake of PFOA and PFOS by V. natans was 41.74 % and 52.31 %, respectively. Notably, PFAS bound to functional proteins (GSTF10), promoting the detoxification of plants. Exposure to PFAS also altered the structure of biofilms by inducing the synthesis of large amounts of polysaccharides and proteins. The diversity and richness of the microbial community within periphytic biofilms changed significantly. These results provide a comprehensive description of the responses of aquatic plants and periphytic biofilms to PFAS and the removal mechanism of PFAS, contributing to the environmental risk assessments and removal of PFAS in aquatic ecosystems.