Daptomycin is a lipopeptide antibiotic that binds and permeabilizes the cell membranes of Gram-positive bacteria. Membrane permeabilization requires both calcium and phosphatidylglycerol (PG) in the target membrane, and it correlates with the formation of an oligomer that likely comprises eight subunits, which are evenly distributed between the two membrane leaflets. In both bacterial cells and model membranes, changes in the fatty acyl composition of the membrane phospholipids can prevent permeabilization. We here used liposomes to study the effect of phospholipids containing oleoyl and other fatty acyl residues on daptomycin activity, and made the following observations: (1) Oleic acid residues inhibited permeabilization when part not only of PG, but also of other phospholipids (PC or cardiolipin). (2) When included in an otherwise daptomycin-susceptible lipid mixture, even 10% of dioleoyl lipid (DOPC) can strongly inhibit permeabilization. (3) The inhibitory effect of fatty acyl residues appears to correlate more with their chain length than with unsaturation. (4) Under all conditions tested, permeabilization coincided with octamer formation, whereas tetramers were observed on membranes that were not permeabilized. Overall, our findings further support the notion that the octamer is indeed the functional transmembrane pore, and that fatty acyl residues may prevent pore formation by preventing the alignment of tetramers across the two membrane leaflets.