The cases of bacterial multidrug resistance are increasing every year and becoming a serious concern for human health. Multidrug efflux pumps are key players in the formation of antibiotic resistance, which transfer out a broad spectrum of drugs from the cell and convey resistance to the host. Efflux pumps have significantly reduced the efficacy of the previously available antibiotic armory, thereby increasing the frequency of therapeutic failures. In gram-negative bacteria, the AcrAB-TolC efflux pump is the principal transporter of the substrate and plays a major role in the formation of antibiotic resistance. In the current work, advanced computer-aided drug discovery approaches were utilized to find hit molecules from the library of biogenic chalcones against the bacterial AcrB efflux pump. The results of the performed computational studies via molecular docking, drug-likeness prediction, pharmacokinetic profiling, pharmacophore mapping, density functional theory, and molecular dynamics simulation study provided ZINC000004695648, ZINC000014762506, ZINC000014762510, ZINC000095099506, and ZINC000085510993 as stable hit molecules against the AcrB efflux pumps. Identified hits could successfully act against AcrB efflux pumps after optimization as lead molecules.Communicated by Ramaswamy H. Sarma.