The number of infections caused by multidrug antibiotic resistance (MDR) species is increasing globally. The efflux pump system, AcrAB-TolC, confers Escherichia coli resistance to many antibiotics and results in poor treatment outcomes. Different rational drug design techniques were employed to search for a safe and effective AcrAB-TolC system inhibitor. Ligand docking was performed to analyze the binding of different ArcB substrates and/or inhibitors in the different AcrAB crystal structure binding sites. The validated docking site using the established docking preferences was used to perform virtual high-throughput screening on a large library of compounds. Domperidone, a known and safe over-the-counter antiemetic drug, was proposed as an effective ArcB inhibitor. Microbiological studies confirmed the computational results and domperidone reversed the resistance to the antibiotics: levofloxacin and ciprofloxacin in the MDR E. coli stains with an effect that surpassed the effect of the known efflux pump inhibitor, reserpine. In addition, it was able to increase both antibiotic effects on susceptible strains. This finding suggests that the antibiotic-domperidone combination can be used clinically to treat infections caused by multidrug-resistant E. coli strains.