Disease caused by nontuberculous mycobacteria (NTM) is reported to increase due to an ageing population and a rise in the proportion of immunosuppressed patients. We did a retrospective cohort study of NTM-disease in the Danish population through a quarter-century to determine the disease burden and trends in annual incidence rates. 524,119 clinical specimens were cultured for mycobacteria from 1991 through 2015 at the International Reference Laboratory of Mycobacteriology in Denmark. Among these, 8,227 NTM strains were identified from 3,462 patients and distributed according to microbiological disease criteria. We observed no increase in NTM disease incidence or proportion of patients with positive NTM cultures during the study period (Quasi-Poisson regression, p = 0.275 and 0.352 respectively). Annual incidence rates were 1.20/105 for definite NTM disease, 0.49/105 for possible NTM disease and 0.88/105 for NTM colonization. The incidence rate of NTM disease was highest in children aged 0-4 years (5.36/105/year), predominantly with cervical Mycobacterium avium complex (MAC) adenitis. Surprisingly, based on more than half a million clinical specimens cultured for mycobacteria in Denmark through 25 years, the NTM disease burden and trend in incidence in the Danish population has not increased opposed to numerous internationals reports.