We aimed at defining profiles of volatile organic compounds in exhaled breath from patients with cystic fibrosis (CF) using a novel real-time mass spectrometry technique. In this prospective matched case-control study, 30 patients with CF, and 30 healthy control subjects were matched one-to-one according to age, gender, and smoking state. We performed exhaled breath analysis by untargeted secondary electrospray ionization-high resolution mass spectrometry (SESI-HRMS). Patients with CF (mean age 26.0 ± 13.0 years) and controls (mean age 27.9 ± 14.0 years) were analyzed using SESI-HRMS. 49 exhaled breath features were found to be altered (p-value < 0.05/q-value < 0.1) in CF patients, in comparison to healthy controls. The two most discriminating features showed a prediction AUROC of 77.1% (95% CI 62.2%-87.8%) with a specificity of 80.0% and a sensitivity of 63.3%. Levels of oxidative stress metabolites such as fatty acids were found to differ significantly between patients with CF and healthy controls. Furthermore, in patients with CF, 11 features correlated with the mucus concentration of Stenotrophomonas maltophilia bacteria. Exhaled breath analysis with SESI-HRMS allows the identification of CF specific compounds in real-time and may trace bacterial strains in affected patients with CF.