Background: Pulmonary tuberculosis is an infectious disease of the lungs caused by Mycobacterium tuberculosis complex (MTB) designated by law in Korea, and accurate diagnosis and urgent treatment are necessary for the maintenance of public health. Recently, nontuberculous mycobacteria (NTM) have been more frequently isolated in respiratory system specimens, which were confused with MTB. We investigated whether the incidence of NTM isolation is increasing in Jeju, Korea. Methods: The results of microbacterial cultures of acid fast bacilli (AFB) from respiratory system specimens were collected at Jeju National University Hospital, Jeju, Korea from 2004 to 2011. The incidences of MTN or NTM isolation were analyzed. Results: A total of 15,484 AFB cultures were performed in 6,281 patients. In 2004, 365 AFB cultures were requested, and the number increased to 1,550 in 2011. However, the culture-positive rate decreased from 18.7% in 2004 to 6.19% in 2011. Among the 573 cultured specimens, 506 MTB (88.3%, mean age of 49.7, male 63.2%) and 72 NTM (12.6%, mean age of 65.8, male 50.0%) were identified. The proportions of NTM isolations were less than 10% until 2009, but increased to 30% after 2010 (P<0.001). M. avium complex (MAC) was the most frequently isolated NTM, followed by M. abscessus. Conclusion: The proportion of NTM isolation is increasing. A clinical diagnosis of pulmonary tuberculosis based on respiratory system specimens should be made with caution, especially in cases of positive AFB smears. (Ann Clin Microbiol 2013;16:13-18)