Pseudomonas aeruginosa is a highly virulent, multidrug-resistant pathogen that causes significant morbidity and mortality in hospitalized patients and is particularly devastating in patients with cystic fibrosis. Increasing antibiotic resistance coupled with decreasing numbers of antibiotics in the developmental pipeline demands novel antibacterial approaches. Here, we tested peptide-conjugated phosphorodiamidate morpholino oligomers (PPMOs), which inhibit translation of complementary mRNA from specific, essential genes in P. aeruginosa. PPMOs targeted to acpP, lpxC, and rpsJ, inhibited P. aeruginosa growth in many clinical strains and activity of PPMOs could be enhanced 2-to 8-fold by the addition of polymyxin B nonapeptide at subinhibitory concentrations. The PPMO targeting acpP was also effective at preventing P. aeruginosa PAO1 biofilm formation and at reducing existing biofilms. Importantly, treatment with various combinations of a PPMO and a traditional antibiotic demonstrated synergistic growth inhibition, the most effective of which was the PPMO targeting rpsJ with tobramycin. Furthermore, treatment of P. aeruginosa PA103-infected mice with PPMOs targeting acpP, lpxC, or rpsJ significantly reduced the bacterial burden in the lungs at 24 h by almost 3 logs. Altogether, this study demonstrates that PPMOs targeting the essential genes acpP, lpxC, or rpsJ in P. aeruginosa are highly effective at inhibiting growth in vitro and in vivo. These data suggest that PPMOs alone or in combination with antibiotics represent a novel approach to addressing the problems associated with rapidly increasing antibiotic resistance in P. aeruginosa.