HT61 and chlorhexidine (CHX) are both putative membrane-active antimicrobials, which non-specifically target the anionic lipids abundant in bacterial membranes. In model systems, the ability of these antimicrobials to partition into lipid monolayers and increase the permeability of lipid bilayers is dependent upon the presence and proportion of anionic lipids such as phosphatidylglycerol. Despite their apparent similarity in membrane affinity, we have found that HT61 and CHX differ in the extent to which they affect membrane integrity. HT61 was found to be capable of severely disrupting the lipid bilayer, resulting in lysis of Staphylococcus aureus membranes and the release of ATP from protoplasts. CHX, by contrast, does not disrupt the lipid bilayer to a sufficiently large degree to result in lysis of the membrane or release of ATP from S. aureus protoplasts. This suggests that although antimicrobials that interact with the membrane often have a common target, the action they have on the membrane may differ widely and may not be the primary mode of action of the antimicrobial.