In this manuscript, we want to review the biochemical and genetic characteristics of the different efflux pumps involved in both intrinsic and acquired multiresistance in non-fermentative Gram-negative bacteria such as Pseudomonas aeruginosa, Acinetobacter baumannii, and Stenotrophomonas maltophilia, as well as the regulation of their expression. Moreover, the clinical impact of the over-expression of these efflux pumps and the investigation developed to define efflux pump inhibitors will be discussed. In this review it will be stated that antimicrobial resistance associated with the over-expression of MDR efflux pumps is widely recognised as a frequent multidrug resistant determinant in nonfermentative Gram-negative bacilli. Moreover, MDR pumps contribute to the intrinsic resistance of these bacterial pathogens. Circumventing the activity of efflux pumps will thus have clear benefits for therapy, since this will increase the susceptibility of nonfermentative Gram-negative bacilli, thereby increasing the therapeutic efficacy of antibiotics used for treating such infections by those pathogens. In addition, it has been shown that the lack of activity of MDR pumps impedes selection of mutants showing high-level antibiotic resistance to antiotics like quinolones or beta-lactams. Thus, besides reducing intrinsic resistance, inhibitors of efflux pumps will reduce the emergence of mutants that acquire antibiotic resistance as the consequence of mutations in MDR-regulatory elements or in other targets. Recent advances on the search for inhibitors of MDR pumps will also be finally discussed.