Microbial communities at the soil-stream-sediment interface provide important ecological functions and services. However, per-and polyfluoroalkyl substances (PFAS), as globally pervasive emerging contaminants, pose a substantial challenge to chemical safety and microbial biodiversity across environmental compartments. This study evaluated the impacts of PFAS contamination in distinct environmental media on bacterial, microeukaryotic, and fungal communities along the soil-stream-sediment continuum. Microbial and PFAS data were collected from six monitoring sites along the lower Beiluo River, in the Loess Plateau, China. PFAS concentrations ranged from 7.50 to 18.46 ng/L in water, 0.21 to 2.49 ng/g dry weight (dw) in soil, and 0.22 to 1.27 ng/g dw in sediment. Microbial assemblages exhibited media-specific patterns; soil and sediment groups shared high similarity in bacterial and microeukaryotic profiles, whereas microeukaryotes and fungi were more similar between water and sediment. Non-metric multidimensional scaling indicated that microbial community distributions were spatially aligned with the PFAS pollution patterns. Pearson and Mantel tests revealed that both traditional long-chain and alternative short-chain PFAS disrupt microbial community stability. Linear mixed-effects models (LMMs) confirmed that bacterial communities displayed greater sensitivity to PFAS than microeukaryotic and fungal communities, as evidenced by more significant perturbations. Redundancy analysis further suggested that both PFAS and nutrients plays a crucial role in shaping microbial community structure across the continuum. This study highlights PFAS impacts on micro-ecosystems and underscores the need for enhanced monitoring and management of riverine systems.