The ability of Pseudomonas aeruginosa to form antibiotic-resistant biofilms is thought to account for the inability of current therapies to resolve bacterial infections in the lungs of patients with cystic fibrosis (CF). We recently described a system in which highly antibiotic-resistant P. aeruginosa biofilms grow on human CF airway epithelial cells, and using this system we showed that enhanced iron release from CF cells facilitates the development of such highly antibiotic-resistant biofilms. Given the positive role for iron in biofilm development, we investigated whether the FDA-approved iron chelators deferoxamine and deferasirox would enhance the ability of tobramycin, the primary antibiotic used to treat CF lung infections, to eliminate P. aeruginosa biofilms. The combination of tobramycin with deferoxamine or deferasirox reduced established biofilm biomass by approximately 90% and reduced viable bacteria by 7-log units. Neither tobramycin nor deferoxamine nor deferasirox alone had such a marked effect. The combination of tobramycin and FDA-approved iron chelators also prevented the formation of biofilms on CF airway cells. These data suggest that the combined use of tobramycin and FDA-approved iron chelators may be an effective therapy to treat patients with CF and other lung disease characterized by antibiotic-resistant P. aeruginosa biofilms.