HT61 is a quinoline-derived antimicrobial, which exhibits bactericidal potency against both multiplying and quiescent methicillin resistant and sensitive Staphylococcus aureus, and has been proposed as an adjunct for other antimicrobials to extend their usefulness in the face of increasing antimicrobial resistance. In this study, we have examined HT61's effect on the permeability of S. aureus membranes and whether this putative activity can be attributed to an interaction with lipid bilayers. Using membrane potential and ATP release assays, we have shown that HT61 disrupts the membrane enough to result in depolarization of the membrane and release of intercellular constituents at concentrations above and below the minimum inhibitory concentration of the drug. Utilizing both monolayer subphase injection and neutron reflectometry, we have shown that increasing the anionic lipid content of the membrane leads to a more marked effect of the drug. In bilayers containing 25 mol % phosphatidylglycerol, neutron reflectometry data suggest that exposure to HT61 increases the level of solvent in the hydrophobic region of the membrane, which is indicative of gross structural damage. Increasing the proportion of PG elicits a concomitant level of membrane damage, resulting in almost total destruction when 75 mol % phosphatidylglycerol is present. We therefore propose that HT61's primary action is directed toward the cytoplasmic membrane of Gram-positive bacteria.