Chronic Pseudomonas aeruginosa lung infection is the most severe complication in cystic fibrosis patients. It is characterized by antibiotic-tolerant biofilms in the endobronchial mucus with zones of oxygen (O2) depletion mainly due to polymorphonuclear leukocyte (PMN) activity. While the exact mechanisms affecting antibiotic effectiveness on biofilms remain unclear, accumulating evidence suggests that the efficiency of several bactericidal antibiotics, such as ciprofloxacin, is enhanced by stimulation of the aerobic respiration of pathogens and that lack of O2 increases their tolerance. Re-oxygenation of O2 depleted biofilms may thus improve susceptibility to ciprofloxacin possibly by restoring aerobic respiration. Methods: We tested such strategy using re-oxygenation of O2-depleted P. aeruginosa strain PAO1 agarose embedded biofilms by hyperbaric O2 treatment (HBOT) enhancing the diffusive supply for aerobic respiration during ciprofloxacin treatment. 3-day-old anoxic biofilms were treated with 1 mg/L ciprofloxacin. The biofilms were further incubated for 90 min ± HBOT (100% O2, 2.8 bar). Results: We demonstrated enhanced bactericidal activity of ciprofloxacin in P. aeruginosa agarose-biofilm during 90 min of HBOT (P < 0.05). In fact, the maximum enhancement of killing by HBOT exceeded 1 log using 1 mg/L of ciprofloxacin. Conclusions: This study demonstrates that biofilm re-oxygenation by HBOT can significantly enhance the bactericidal activity of ciprofloxacin on P. aeruginosa during 90 min of incubation. Combining ciprofloxacin treatment with HBOT thus clearly has potential to improve the treatment of P. aeruginosa biofilm infections.