The rise in antimicrobial resistant bacteria is limiting the number of effective treatments for bacterial infections.andare two of the pathogens with the highest prevalence of resistance, and with the greatest need for new antimicrobial agents. Combinations of antimicrobial peptides (AMPs) and antibiotics that display synergistic effects have been shown to be an effective strategy in the development of novel therapeutic agents. In this study, we investigated the synergy between the AMP LL-37 and various classes of antibiotics againstandstrains. Of the six antibiotics tested (ampicillin, tetracycline, ciprofloxacin, gentamicin, aztreonam, and polymyxin B (PMB)), LL-37 displayed the strongest synergy againstMG1655 andPAO1 laboratory strains when combined with PMB. Given the strong synergy, the PMB + LL-37 combination was chosen for further examination where it demonstrated synergy against multidrug-resistant and clinicalisolates. Synergy of PMB + LL-37 towards clinical isolates ofvaried and showed synergistic, additive, or indifferent effects. The PMB + LL-37 combination treatment showed significant prevention of biofilm formation as well as eradication of pre-grownandbiofilms. Using thewax worm model, we showed that the PMB + LL-37 combination treatment retained its antibacterial capacities in vivo. Flow analyses were performed to characterize the mode of action. The results of the present study provide proof of principle for the synergistic response between LL-37 and PMB and give novel insights into a promising new antimicrobial combination against gram-negative planktonic and biofilm cells.