The rates of pulmonary colonization and disease due to nontuberculous mycobacteria (NTM) appear to be increasing globally, but diversity of species recovered as well as clinical relevance of NTM isolates differ considerably by geographic region. The first nationwide study of isolation frequency and clinical significance of NTM in Serbia included all patients with respiratory specimens yielding a positive NTM culture over the six-year period, 2010-2015. We analyzed trends in annual NTM isolation and NTM pulmonary disease (PD) incidence rates, with NTM PD cases defined in accordance with microbiological criteria established by the American Thoracic Society/Infectious Diseases Society of America (ATS/IDSA). 777 pulmonary NTM isolates were collected from 565 patients, of whom 126 (22.3%) met the ATS/IDSA criteria. The annual NTM isolation and NTM PD incidence rates per 100,000 changed over 2010-2015 from 0.9 to 1.6 (p = 0.1746) and from 0.18 to 0.48 (p = f0.0040), respectively. Both isolation and disease rates increased considerably with age, while higher NTM PD rates were also associated with residence in urbanized areas. Diversity of NTM species isolated was shown to be region-specific, with M. xenopi as the most prevalent species (17.3%), and increasing isolation rates of rapid growing mycobacteria (RGM) (p = 0.0072). M. xenopi was also the most common cause of NTM PD (28.6%), followed by RGM (27.8%). With 73% clinically relevant isolates, M. abscessus was identified as the most clinically relevant NTM species. While NTM PD obviously remains a rare disease in Serbia, the overall results justify recognition of NTM as pathogens of rising importance, and require further characterization of their epidemiology in the country.