(1) Background: Cystic fibrosis (CF) is characterized by chronic pulmonary inflammation and persistent bacterial infections. is among the main opportunistic pathogens causing infections in CF. is able to form a biofilm, decreasing antibiotic permeability. LOX, a lipoxygenase enzyme, is a virulence factor produced by and promotes its persistence in lung tissues. The aim of this study is to evaluate if antibiotics currently used for aerosol therapy in CF are able to interfere with the production of lipoxygenase from open isolates of from patients with CF. (2) Methods: Clinical isolates of from patients with CF were grown in Luria broth (LB). Minimum inhibitory concentration (MIC) was performed and interpreted for all isolated strains according to the European Committee on Antimicrobial Susceptibility Testing (EUCAST) guidelines. We selected four antibiotics with different mechanisms of action: aztreonam, colistin, amikacin, and levofloxacin. We used human pulmonary epithelial NCI-H929 cells to evaluate LOX activity and its metabolites according to antibiotic action at increasing concentrations. (3) Results: there is a correlation between LOX secretion by clinical isolates of and biofilm production. Levofloxacin exhibits highly significant inhibitory activity compared to the control. Amikacin also exhibits significant inhibitory activity against LOX production. Aztreonam and colistin do not show inhibitory activity. These results are also confirmed for LOX metabolites. (4) Conclusions: among the evaluated antibiotics, levofloxacin and amikacin have an activity on LOX secretion.