Staphylococcus aureus is a serious opportunistic bacterial pathogen responsible for multidrug resistant hospital- or community-acquired infections with significant morbidity and mortality worldwide. Distressingly, this ‘Superbug’ has developed resistance to quaternary ammonium compounds (QACs) which are widely used in medical and industrial settings as disinfectants and antiseptics. A main factor in the development of resistance to QACs is the expression of drug efflux pumps such as QacA, which is the most prevalent plasmid-encoded multidrug efflux pump found in clinical S. aureus isolates. QacA, is able to confer resistance to >30 structurally dissimilar monovalent and bivalent cationic antimicrobial agents. However, QacA structure-function relationships have not been fully resolved. QacA is comprised of 14 transmembrane segments (TMS) and TMS 12 has been suggested to be a component of the bivalent cation-binding region. This study aimed to delineate the functional importance of TMS 12. Toward this end, 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 assessed. Western blotting analyses showed all QacA mutants were expressed at levels close to wild-type, implying that cysteine substitution in QacA did not affect protein expression. Minimum inhibitory concentration assays with representative antimicrobial compounds revealed G361, G379 and S387 had a significant impact (≤50% reduction) on QacA resistance capacity for at least one of the bivalent cationic substrates, indicating the importance of these residues in the interaction with these substrates. Our results confirm the functional interplay between TMS 12 of QacA and bivalent cationic substrates. Further fluorimetric transport assays and binding studies are in progress to determine as to whether TMS 12 directly involves in the substrate translocation and/or binding process. Elucidating the detailed role of TMS 12 is an imperative step towards the ultimate goal of translating the findings into development of novel antimicrobials/inhibitors that combat QacA-mediated resistance.