Background: Pseudomonas aeruginosa is a major pathogen of the chronic lung infections in cystic fibrosis (CF) patients. These persistent bacterial infections are characterized by bacterial aggregates with biofilm-like properties and are treated with nebulized or intravenous tobramycin in combination with other antibiotics. However, the chronic infections are close to impossible to eradicate due to reasons that are far from fully understood. Recent work has shown that re‑oxygenation of hypoxic aggregates by hyperbaric oxygen (O ₂ ) treatment (HBOT: 100% O ₂ at 2.8 bar) will increase killing of aggregating bacteria by antibiotics. This is relevant for treatment of infected CF patients where bacterial aggregates are found in the endobronchial secretions that are depleted of O ₂ by the metabolism of polymorphonuclear leukocytes (PMNs). The main objective of this study was to investigate the effect of HBOT as an adjuvant to tobramycin treatment of aggregates formed by P. aeruginosa isolates from CF patients. Methods: The effect was tested using a model with bacterial aggregates embedded in agarose. O ₂ profiling was used to confirm re‑oxygenation of aggregates. Results: We found that HBOT was able to significantly enhance the effect of tobramycin against aggregates of all the P. aeruginosa isolates in vitro. The effect was attributed to increased O ₂ levels leading to increased growth and thus increased uptake of and killing by tobramycin. Conclusions: Re‑oxygenation may in the future be a clinical possibility as adjuvant to enhance killing by antibiotics in cystic fibrosis lung infections.