Recent reports indicated an increase of nontuberculous mycobacteria (NTM) in cystic fibrosis (CF) patients. However, it is still a matter of discussion whether criteria for diagnosis of NTM pulmonary infection established by the American Thoracic Society (ATS) are applicable for CF patients. We determined incidence and prevalence of NTM in CF patients and non-CF patients without HIV infection, and validity of ATS criteria for CF patients. Over a period of 2 years, 1,251 respiratory samples were investigated for mycobacteria from 91 CF and 162 non-CF patients. For all patients with NTM recovery, we reviewed clinical charts and determined outcome for up to 2 years. Incidence and prevalence for repeated recovery of NTM were higher in CF patients, but not significantly. CF patients with repeated recovery of NTM met clinical and bacteriological ATS criteria, but radiographic criteria were not met. Treated CF patients showed favorable clinical outcomes, as opposed to untreated patients. We propose a modified definition for diagnosis and hence treatment of NTM pulmonary infection in CF patients.