A novel series of 4-substituted 2-naphthamide derivatives were designed, synthesized and evaluated for their biological activity. In particular, the ability of the compounds to potentiate the action of antibiotics, to inhibit Nile Red efflux and to target AcrB specifically was investigated. The results indicated that most of the 4-substituted 2-naphthamide derivatives were able to synergize with the antibiotics tested, and inhibit Nile Red efflux by AcrB in the resistant phenotype. Subsequent exclusion of compounds with off target effects such as outer- or inner membrane permeabilization identified compounds 7c, 7g, 12c, 12i and 13g as efflux pump inhibitors (EPIs). Particularly, compounds 7c, 7g and 12i were found to be the most potent EPIs, which synergized with the two substrates tested at lower concentrations than that of parent A3, demonstrating an improvement in potency as compared to A3. Additionally, when the outer membrane of E. coli was permeabilized, compound 12c displayed a huge increase in efficacy and was able to synergize with erythromycin at a concentration that was 16 times lower than that of the parent A3. Hence we were able to design and synthesize compounds that displayed significant increase in efficacy as EPIs against AcrB.