This study investigated the phytotoxicity, physiological effects and bioaccumulation of perfluorohexane sulfonic acid (PFHxS) in two Australian native plants, Iseilema membranaceum and Enchylaena tomentosa, grown in natural field soil and OECD artificial soil under glasshouse conditions. Soils were spiked with PFHxS at concentrations ranging from 0 to 1000 mg/kg, and plants were monitored for up to nine weeks. Seedling emergence was not significantly affected up to 100 mg/kg in field soil and 250 mg/kg in OECD soil. However, sub-lethal endpoints such as shoot length and biomass were more sensitive, with EC50 values as low as 51.3 mg/kg for I. membranaceum seedling emergence and 139.6 mg/kg for E. tomentosa shoot height. Total chlorophyll and Malondialdehyde (MDA) content in I. membranaceum decreased with increasing concentration of PFHxS whereas those increased in E. tomentosa. Peroxidase activity decreased in both plants as PFHxS concentration rose, yet no significant difference was observed compared to controls. A concentration-dependant bioaccumulation pattern of PFHxS was noted, with roots accumulating more PFHxS than shoots. Notably, I. membranaceum roots showed higher accumulation of PFHxS at soil concentration of 100 mg/kg. Transmission electron microscopy (TEM) coupled with EDS revealed PFHxS localization in the endoplasmic reticulum and phloem parenchyma cells, suggesting intracellular transport and retention mechanisms. These findings offer new insights into PFHxS uptake and effects in arid-adapted native plants, contributing critical data for terrestrial ecological risk assessments.