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Multidrug resistance, whereby pathogens develop resistance to several antimicrobial agents, is a large and growing public health concern globally. In order to combat its continued spread, a better understanding of resistance mechanisms is necessary. One recurring mechanism of multidrug resistance is through the activities of multidrug efflux pumps. These pumps remove a variety of potentially noxious compounds from inside the cells of bacteria and other infectious organisms. In this study, I have looked at multiple families of efflux pumps in an effort to answer the most pressing mechanistic questions in each family using both computational and experimental methods. The Multidrug and Toxic Compound Extrusion (MATE) family is one of the most recently recognized and least well characterized. Using the high-resolution structures available, we reinterpret existing crystallographic data to reveal the existence of a novel sodium-binding site. We observe that this site is widely conserved in the family, and our Molecular Dynamics simulations show that it has mechanistic importance. From this evidence, we reason that sodium is used as the cotransport ion that drives secondary active transport in the MATE family despite prior conflicting experimental evidence on this ion's identity. The Major Facilitator Superfamily (MFS) is the largest family of drug transporters, but until recently none of its members have been structurally elucidated in both inward- and outward- facing states. These are now available for MdfA, and we have conducted simulations to explore how its ligands transfer from the lipid environment to be engaged with these two states. By observing lipid interactions with both the protein and its ligands, we suggest a pathway by which the ligands can travel from the membrane to bound positions. Finally, the Resistance-Nodulation-Cell Division (RND) superfamily contains one of the most well-studied drug efflux complexes: AcrAB-TolC. Though numerous structures exist for this multi-protein assembly, none have been obtained without use of detergents that displace natural lipids which may be needed to support key allosteric states. To better characterize lipid interactions with this complex, we developed and optimized a detergent-free purification protocol for structural analysis by cryogenic election microscopy. To explore its mobility within the cellular milieu, we also attempted to cross-link the complex with the peptidoglycan layer in an effort to identify points of contact with the pump.