Staphylococcus aureus is a major bacterial pathogen responsible for multidrug resistant hospital- or community-acquired infections with significant morbidity and mortality throughout the world. Alarmingly, this ‘Superbug’ has developed resistance to quaternary ammonium compounds (QACs) which are widely used as disinfectants and antiseptics in medical, industrial and household settings. A main underlying factor in this resistance is the expression of drug efflux pumps such as QacA, which is by far the most prevalent plasmid-encoded multidrug efflux pump found in clinical S. aureus isolates. QacA is able to confer resistance to >30 structurally different monovalent and bivalent cationic antimicrobial agents. To date, QacA structure-function relationships have not been fully resolved. QacA is comprised of 14 transmembrane segments (TMS) and TMS 12 has been proposed to be a component of the bivalent cation-binding region. To delineate the functional importance of TMS 12, 30 amino acid residues within putative TMS 12 and its flanking region were individually substituted with cysteine and the impact of these substitutions on QacA-mediated resistance and efflux activities assessed. Western blotting analyses showed all QacA mutants were expressed at levels similar to wild-type. Resistance profiling identified three residues in the target region that when mutated produce a decreased resistance capacity to at least one of the six representative QacA substrates, indicating the importance of these residues in interaction with specific substrates. Fluorimetric transport assays found two residues with impaired ethidium efflux activity suggesting their involvement in QacA substrate translocation process. Our results confirm the functional interplay between TMS 12 of QacA and bivalent cationic substrates. Further binding studies are underway to determine as to whether functionally important residues in QacA directly involve in substrate binding process. The emerging picture of detailed structure and function of QacA is an imperative step towards the ultimate goal of translating the findings into development of novel antimicrobials/inhibitors for countering QacA-mediated resistance.