This study evaluated PFAS contamination risks associated with biosolids land application in the New York State (NYS) Great Lakes basin. Environmental samples collected from soil, surface water, and vegetation at several sites previously treated with biosolids showed the presence of PFAS at trace levels. No exceedances of EPA maximum contaminant levels (MCLs) were detected in surface water samples. However, significant bioaccumulation of PFHxS and N-EtFOSAA was observed in the collected plant tissues. A complementary greenhouse study was conducted to assess PFAS migration and fractionation in controlled plant-biosolids-soil systems. Short-chain PFAS exhibited greater mobility and plant bioavailability, whereas longer-chain PFAS remained strongly bound in soil. Plant cultivation altered PFAS fractionation, enhancing short-chain PFAS leachability. Microbial community analysis showed that biosolids amendment and PFAS exposure significantly affected soil microbial diversity and structure. These findings highlight the complexity of PFAS behavior in environmental systems and the importance of integrated management strategies to minimize environmental risks and safeguard public health.