Background: Patients with cystic fibrosis have increased morbidity/mortality due to chronic respiratory infections, which primarily originate from the environment. Infection prevention and control emphasize the importance of cleaning and disinfection of respiratory devices, however, there is a paucity of guidance on toothbrush hygiene, which have been shown to be a source of cystic fibrosis (CF) pathogens. Methods: This study examined steam disinfection of toothbrushes contaminated with clinically significant CF isolates (n = 80; Gram positive = 33; Gram negative = 32, and non-tuberculous mycobacteria = 6) and yeasts (n = 9), as well as oral streptococci (n = 26) and environmental Pseudomonas aeruginosa (n = 12). Results: Steam disinfection eradicated all organisms tested, as well as all organisms in CF sputum applied to toothbrushes. Conclusions: Steam disinfection offers a relatively simple, cheap and available method of eliminating non-spore-forming CF pathogens on toothbrushes. Toothbrushes should be thoroughly rinsed after each use before steam disinfection, to remove plaque, epithelial cells, and residual toothpaste. Toothbrushes should be steam disinfected after each use employing a baby bottle steam disinfector, adhering to manufacturers' operating instructions and stored in the disinfector until next used within 12 to 24 hours. Toothbrushes should be replaced every 3 to 4 months, or sooner if the bristles look worn out, as well as every time a pulmonary exacerbation occurs or every time the patient is treated for a pulmonary/throat infection. Steam disinfection of toothbrushes is crucial when the patient is undergoing eradication regimes for P. aeruginosa and methicillinresistant Staphylococcus aureus, so that the patient does not become reinfected from this source, thereby aiding eradication and enhancing patient safety.