Intractable formation of biofilm by and infection with the opportunistic pathogen Pseudomonas aeruginosa are hallmarks of cystic fibrosis (CF). Lactoferrin, an innate immunity protein, has recently been shown to inhibit the formation of P. aeruginosa biofilm. Partial cleavage of lactoferrin by the proteases neutrophil elastase and Pseudomonas elastase has previously been described in CF. Here, we show that cathepsins in CF secretions are responsible for complete and rapid cleavage of lactoferrin. We demonstrate that levels of lactoferrin in P. aeruginosa-positive sputum samples are decreased when corrected for inflammatory burden and that P. aeruginosa-positive sputum samples have significantly higher cathepsin activity and significantly reduced ability to inhibit formation of biofilm, compared with P. aeruginosa-negative sputum samples. We also show that cleavage of lactoferrin by cathepsin results in loss of both its microbicidal and antibiofilm activity. Loss of such a vital innate immunity protein clearly has important implications for the pathogenesis of chronic P. aeruginosa lung infection in patients with CF.