Perfluorohexanesulfonic acid (PFHxS) is one of the persistent organic pollutants that has been listed in Annex A of the Stockholm Convention. It has gained increasing attention in recent years due to its toxic effects. In this study, multispecies bioassays were conducted to determine the ecological toxic effects of PFHxS, including plants, soil invertebrates, and soil microorganisms, which indicated the EC10/NOEC values ranged from 2.9 – 250 mg/kg. The toxic effects were observed for plant seed germination, shoot and root length, chlorophyll content, mortality of earthworm, and microbial alternations. The various toxic endpoints were modelled using logistic models to derive the effective values. The species sensitivity distributions (SSD) were employed to estimate the ecological threshold levels for PFHxS contamination in soils using toxicity results from both literature and this study. The HC5 (hazardous concentration for 5% of the species being impacted) were calculated. The results obtained in this study can be useful in risk assessment processes to better understand the toxic effects of PFHxS and minimize any uncertainties using combined screening values with PFOS.