Bacterial multidrug resistance poses an ever increasing threat to human health. Integral to this are multidrug resistance transporters, which are membrane proteins that have the ability to expel a broad spectrum of chemically dissimilar drugs out of the cell in an energy dependent manner, using either ATP hydrolysis or the ion motive force. These efflux proteins fall into one of five families: Major Facilitator Superfamily (MFS), ATP binding cassette (ABC), Resistance-Nodulation-Division (RND), Small Multidrug Resistance (SMR) and Multidrug And Toxic compound Exporters (MATE). Structural information on these classes of proteins is limited due to problems inherent to isolating and crystallising integral membrane proteins. Thus, we have combined bioinformatics, genetics, biophysical and biochemical tools to examine the multidrug resistance capacity of the QacA efflux protein from Staphylococcus aureus. QacA is a MFS family membrane protein, containing 14 transmembrane segments (TMS), that confers resistance to more than 30 different cationic, lipophilic antimicrobial compounds commonly used as antiseptics and disinfectants. Comparison of structures and the efflux mechanism of QacA with the 12 TMS MdfA MFS multidrug resistance protein from Escherichia coli has provided insight into how secondary transporters deal with compounds of different valencies. Additionally, construction and analysis of mutants, including chimeras between these two proteins, has identified a core 12 TMS structure required for active transport by both proteins.