The drug/proton antiporter AcrB, which is part of the major efflux pump AcrABZ-TolC in Escherichia coli, is the paradigm transporter of the resistance-nodulation-cell division (RND) superfamily. Despite the impressive ability of AcrB to transport many chemically unrelated compounds, only a few of these ligands have been co-crystallized with the protein. Therefore, the molecular features that distinguish good substrates of the pump from poor ones have remained poorly understood to date. In this work, a thorough in silico protocol was employed to study the interactions of a series of congeneric compounds with AcrB to examine how subtle chemical differences affect the recognition and transport of substrates by this protein. Our analysis allowed us to discriminate among different compounds, mainly in terms of specific interactions with diverse sub-sites within the large distal pocket of AcrB. Our findings could provide valuable information for the design of new antibiotics that can evade the antimicrobial resistance mediated by efflux pump machinery.