The increasing emergence of multidrug-resistant bacteria is a major health threat worldwide. Staphylococcus aureus is one of the most serious human pathogens which is responsible for burgeoning multidrug-resistant hospital- or community-acquired infections with significant morbidity and mortality globally. S. aureus utilises a number of mechanisms to circumvent the effects of antimicrobials, one of these is the export of antimicrobial agents through the activity of membrane-embedded multidrug efflux pump proteins. QacA is a staphylococcal efflux pump that exports multiple antimicrobial compounds, many commonly used as antiseptics and disinfectants, such as benzalkonium and chlorhexidine. QacA is a 14-TM (transmembrane) helix protein that belongs to the major facilitator superfamily (MFS) of transport proteins. The transport process used by MFS proteins, including QacA, depends on distinct subsets of key residues that interact with each specific substrate, the first step in coordinating substrate passage across the membrane. Therefore, a detailed understanding of the structural and functional features of QacA is a prerequisite for developing a strategy to overcome/block its efflux activity. In this study, homology modelling and molecular docking methods were employed in combination with extensive site-directed cysteine-scanning mutagenesis data. The results identified putative multidrug binding pockets of QacA as well as hotspot regions lining the QacA central cavity that potentially interact with benzalkonium and chlorhexidine. These regions are located at different depths in the membrane and are formed by a cluster of functionally-important residues from multiple TM helices. Overall, a blend of microbiological, biochemical, and bioinformatic data has provided new insights into the architectural features of QacA drug binding pockets will be indispensable for rationale efflux pump inhibitor development.