Since we first recognized the regular presence of gaseous hydrogen cyanide, HCN, in the headspace of plate cultures of the bacterium Pseudomonas aeruginosa, PA, derived from sputum of cystic fibrosis, CF, patients, and following crucial ion chemistry research that allowed accurate quantification of gaseous HCN by selected ion flow tube mass spectrometry, we have carried out many further in vitro and in vivo studies. We have measured HCN in the headspace of various PA culture types, planktonic and biofilm, significant numbers of genetically identified PA strains together with studies of HCN in the mouth-exhaled and nose-exhaled breath of healthy children and adults and those with CF. The major findings are: (i) virtually all strains of PA release HCN when cultured in vitro, as shown by the investigation of more than 150 genetically differentiated strains, both mucoid and non-mucoid. (ii) HCN is present in the mouth-exhaled breath of adults and children, but is at lower concentrations in children. Its concentration is below the detection limit in nose-exhaled breath of healthy people. (iii) HCN is present in both mouth-exhaled and nose-exhaled breath of patients with CF, suggesting the presence of PA in the lower airways as indicated by clinical microbiological cultures. With confirmation of these findings by further research and clinical trials, nose-exhaled breath HCN measurements could be an additional diagnostic tool to detect the early presence of PA in the lower airways and a non-invasive monitor to enhance the likelihood of its eradication.