Bacterial resistance to antibiotics has been long recognized as a priority to address for human health. Among all micro-organisms, the so-called multi-drug resistant (MDR) bacteria, which are resistant to most, if not all drugs in our current arsenal, are particularly worrisome. The World Health Organization has prioritized the ESKAPE (Enterococcus faecium,Staphylococcus aureus,Klebsiella pneumoniae,Acinetobacter baumannii,Pseudomonas aeruginosaandEnterobacterspecies) pathogens, which include four Gram-negative bacterial species. In these bacteria, active extrusion of antimicrobial compounds out of the cell by means of ‘molecular guns’ known as efflux pumps is a main determinant of MDR phenotypes. The resistance-nodulation-cell division (RND) superfamily of efflux pumps connecting the inner and outer membrane in Gram-negative bacteria is crucial to the onset of MDR and virulence, as well as biofilm formation. Thus, understanding the molecular basis of the interaction of antibiotics and inhibitors with these pumps is key to the design of more effective therapeutics. With the aim to contribute to this challenge, and complement and inspire experimental research,in silicostudies on RND efflux pumps have flourished in recent decades. Here, we review a selection of such investigations addressing the main determinants behind the polyspecificity of these pumps, the mechanisms of substrate recognition, transport and inhibition, as well as the relevance of their assembly for proper functioning, and the role of protein–lipid interactions. The journey will end with a perspective on the role of computer simulations in addressing the challenges posed by these beautifully complex machineries and in supporting the fight against the spread of MDR bacteria.