Antimicrobial resistance in the human pathogen Neisseria gonorrhoeae, the causative agent of gonorrhoea, has reached an alarming level, severely impacting effective treatment. A key mechanism this bacterium uses to achieve multidrug resistance is the expulsion of structurally different antimicrobials from the cell before they reach their target. The primary system that achieves this is the the MtrCDE efflux complex, where the integral inner membrane component of this system, the MtrD protein, dictates substrate selection and binding. The MtrD crystal structure has revealed that, like its homologues, the protein possesses an open cleft containing putative access and deep binding pockets proposed to interact with substrates. A combination of long-timescale molecular dynamics simulations and docking studies together with site-directed mutagenesis of selected residues was used to analyse the structural basis of MtrD:substrate interactions. Of the MtrD mutants generated, only one retained a wild-type resistance profile, while others showed reduced resistance to distinct antimicrobial compounds. Docking studies of eight MtrD substrates confirmed that many of these identified residues play important non-specific roles in binding to these substrates. Long timescale molecular dynamics simulations of MtrD with its substrate progesterone showed spontaneous binding of the substrate to the access pocket of the binding cleft and its subsequent penetration into the deep binding pocket allowing the permeation pathway for a substrate through this important resistance mechanism to be identified. These studies provide a detailed picture of the interaction of MtrD with substrates that can be used as a basis for rational antibiotic and inhibitor design.