Perfluoroalkyl substances (PFAS) are ubiquitous and persistent environmental contaminants, yet knowledge of their biological effects and mechanisms of action is limited. The highest aqueous PFAS concentrations are found in areas where bacteria are relied upon for functions such as nutrient cycling and contaminant degradation, including fire-training areas, wastewater treatment plants, and landfill leachates. This research sought to elucidate one of the mechanisms of action of PFAS by studying their uptake by bacteria and partitioning into model phospholipid bilayer membranes. PFAS partitioned into bacteria as well as model membranes (phospholipid liposomes and bilayers). The extent of incorporation into model membranes and bacteria was positively correlated to the number of fluorinated carbons. Furthermore, incorporation was greater for perfluorinated sulfonates than for perfluorinated carboxylates. Changes in zeta potential were observed in liposomes but not bacteria, consistent with PFAS being incorporated into the phospholipid bilayer membrane. Complementary to these results, PFAS were also found to alter the gel-to-fluid phase transition temperature of phospholipid bilayers, demonstrating that PFAS affected lateral phospholipid interactions. This investigation compliments other studies showing that sulfonated PFAS and PFAS with more than seven fluorinated carbons have a higher potential to accumulate within biota than carboxylated and shorter-chain PFAS.