We present the real-time monitoring of hydrogen cyanide (HCN) production from Pseudomonas aeruginosa (P. aeruginosa) strains in vitro, using laser-based photoacoustic spectroscopy. Simultaneously, the production of ammonia (NH3) was measured, and the influence of different factors (e.g. the medium, temperature and antibiotics treatment) was assessed. Both reference strains and clinical isolates of patients with CF were studied, and compared to other pathogens commonly present in lungs/airways of CF patients. Hydrogen cyanide production starts to rise as soon as P. aeruginosa bacteria reach the stationary phase ((9.0-9.5) × 10(9) colony forming units, CFUs), up to concentrations of 14.5 microliters per hour (µl h(-1)). Different strains of P. aeruginosa produced HCN to varying degrees, and addition of tobramycin strongly reduced HCN production within 2 h from application. Burkholderia cepacia also produced HCN (up to 0.35µl h(-1) in 9.0 × 10(9) CFU) while other pathogens (Aspergillus fumigatus, Stenotrophomonas maltophilia, Mycobacterium abscessus) did not produce detectable levels. Our study reveals for the first time a broad overview of the dynamics of the HCN production in vitro.