Abstract: Waste-derived soil amendments and fertilizers contain high levels of nitrogen and per- and polyfluoroalkyl substances (PFAS), which are of great environmental concern. Co-contamination of nitrate and PFAS in agricultural areas has been confirmed. Denitrification is a key process controlling nitrate transport and transformation in groundwater. However, PFAS are toxic to bacteria. They can change the abundance and composition of groundwater microbial communities, thus affecting denitrification. Currently, the effects of PFAS stress on nitrate reduction in groundwater and the associated microbial response mechanisms remain unclear. Perfluorooctane sulfonate (PFOS) is frequently detected in groundwater in agricultural areas. This study selected PFOS as a typical PFAS. Batch experiments were conducted to investigate the effects of PFOS on denitrification. The mechanisms were analyzed using multiple indicators, including water quality parameters, microbial growth, carbon source metabolism capacity, microbial community structure, and denitrifying enzyme activities. The results showed that low-concentration PFOS (0.1 mg/L) promoted denitrification, while high-concentration PFOS (10 mg/L) significantly inhibited denitrification. The inhibitory effect was especially obvious on the nitrite reduction step. Further mechanism analysis revealed that PFOS regulated bacterial growth and the activities of denitrifying enzymes (nitrate reductase NAR and nitrite reductase NIR), thereby jointly affecting denitrification efficiency. PFOS also drove changes in microbial community structure, leading to enrichment of incomplete denitrifiers and decline of complete denitrifiers. This may increase the potential risk of N₂O emissions. This study reveals the effects and mechanisms of PFOS on groundwater denitrification. It provides a theoretical basis for risk assessment and ecological management of combined pollution in agricultural groundwater.