A key mechanism used by bacteria to achieve resistance to multiple compounds simultaneously is through the expulsion of structurally different antimicrobials from the cell before they reach their target. There is also increasing evidence that efflux pumps promote the formation of biofilms, an adaptation that influences survival in the presence of antimicrobial agents. Through these various efflux pump associated mechanisms, enhanced bacterial survival provides a window within which further adaptive resistance mechanisms can be acquired, either via mutational selection, or plasmids that carry drug resistance genes. Therefore, it is of fundamental importance to understand exactly how these multidrug efflux proteins recognise and export multiple compounds, to enable the rational design of drugs that are not recognised by these systems or compounds that can act as efflux pump inhibitors (EPIs) that obstruct their action and potentiate the activity of currently ineffectual antimicrobials. We have focussed our recent studies on two multidrug efflux systems, the QacA and MtrCDE transport system, that are present in different bacterial pathogens. Construction and analysis of a large set of site-directed mutants has identified residues that potentially comprise the drug binding site(s). This has been explored further using a combination of long-timescale molecular dynamics simulations and docking studies allowing the proposal of potential substrate permeation pathways and the energy mechanism used to power drug transport. Resistance and checkerboard assays with combinations of QacA substrates and potential EPIs, have identified the first set of compounds that potentiate the action of the QacA drug efflux pump and provided the basis for further EPI development.