Pseudomonas aeruginosa airway infection is common in cystic fibrosis (CF), a disease also characterized by abundant extracellular DNA (eDNA) in the airways. The eDNA is mainly derived from neutrophils accumulating in the airways and contributes to a high sputum viscosity. The altered environment in the lower airways also paves the way for chronic P. aeruginosa infection. Here, we show that mice with P. aeruginosa airway infection have increased survival and decreased bacterial load after topical treatment with DNase. Furthermore, DNA from the sputum of CF patients showed increased bactericidal activity after treatment with DNase ex vivo. Both degraded DNA of neutrophil extracellular traps (NETs) and genomic DNA degraded by serum, acquired bactericidal activity against P. aeruginosa. In vitro, small synthetic DNA-fragments (<100 base pairs) but not large fragments nor genomic DNA, were bactericidal against Gram-negative but not Gram-positive bacteria. The addition of divalent cations reduced bacterial killing, suggesting that chelation of divalent cations by DNA results in destabilization of the lipopolysaccharide (LPS) envelope. This is a novel antibacterial strategy where fragmentation of eDNA and DNA-fragments can be used to treat P. aeruginosa airway infection.