We embarked on a program to identify efflux pump inhibitors in Pseudomonas aeruginosa as a way of potentiating the activity of the fluoroquinolone antibacterial agent levofloxacin. We screened our synthetic compound and fermentation extract collection in the presence of levofloxacin using specifically engineered strains of P. aeruginosa that overexpressed each of the known pumps, defined the term MPC8 as the minimum concentration (µg/mL) of inhibitor required to decrease (potentiate) the MIC of levofloxacin 8-fold, and identified compound 1 (MC-207,110), a low molecular weight dipeptide amide, as a broad-spectrum efflux pump inhibitor in P. aeruginosa with minimal intrinsic antibacterial activity (MIC > 512 µg/mL) and ability to potentiate levofloxacin 8-fold at 10 µg/mL. We then conducted an extensive medicinal chemistry program to optimize the biological and physicochemical properties of this lead compound, yielding analogues such as 5 (stable to serum proteases and as potent as compound 3) and 13 (retaining broad-spectrum pump inhibitory characteristics). Compound 5, when administered in combination with levofloxacin in a murine neutropenic thigh model against P. aeruginosa PAM 1032, resulted in a 3-log reduction in colony forming units for approximately 4 h, demonstrating in vivo efficacy. In summary, we have described the first known class of broad-spectrum efflux pump inhibitors in P. aeruginosa.