Due to wide use of perfluoroalkyl substances (PFASs) (e.g., in metal-plating, in fire-fighting foam, lubricants) and their resistance to degradation, they occur widely in the environment. The aim of this study was to estimate the environmental risk resulting from the presence of PFASs in the Gulf of Gdansk. Therefore, 17 PFASs concentrations were determined using ultra performance liquid chromatography with tandem mass spectrometry detection (UPLC-MS/MS). Additionally, sediment ecotoxicity was investigated. The results of the chemical analysis were used to asses environmental risk of PFASs. In samples collected around discharge collectors from a wastewater treatment plant and the Vistula mouth, Σ17PFASs values were 0.00403 ÷ 40.6 and 0.509 ÷ 614 ng/g d.w., respectively. In samples collected around discharge collectors, PFHxA, PFPeA, PFHpA, and PFOA were dominating, while at the Vistula River mouth, PFHxS, PFDS, and PFBS were prevalent. For most sediments, no toxic effect was observed in the toxicity tests with Heterocypris inconguens and Aliivibrio ficsheri. There was no observed correlation between the PFASs level and their ecotoxicity. Generally, the results of environmental risk assessment indicate that the PFASs would not generate high impact on the aquatic life (five water samples have shown medium risk related to PFBS and PFDoA).