Background: Mycobacterial infection is a problem throughout the world along with the increase of immunocompromised patients. For this reason, there have been many methods for faster and more accurate diagnosis. In this study, we evaluated several laboratory methods for mycobacterial infection. Methods: From January to December 2009, 635 specimens were cultured with mycobacteria growth indicator tube (MGIT) and Ogawa media. Polymerase chain reaction (PCR) was performed with the AdvanSure tuberculosis (TB)/non-tuberculosis mycobacterium (NTM) real-time PCR Kit (LG Life Sciences, Seoul, Korea). The 69 samples showing positive culture results were identified with the AdvanSure Mycobacteria Genotyping Chip Kit (LG Life Science, Seoul, Korea). Results: Sixty-nine (10.9%) out of 635 samples showed positive results for mycobacterial culture. Among the 635 samples, 64 were positive in MGIT, but only 42 were positive in Ogawa media. Of the 635 samples, 607 (95.6%) showed the same results between MGIT and Ogawa and the results of 579 (95.4%) were also consistent with the TB/NTM real-time PCR results. However, in the case of NTM, only one (1/24, 4.2%) was positive in PCR. In the Mycobacteria genotyping chip analysis, the most frequently identified NTM species in descending order were M. avium, M. intracellulare, M. chelonae and M. abscessus. Conclusion: Culturing with a combination of MGIT and Ogawa is recommended to increase the recovery rate of mycobacteria. Although PCR missed a reasonable number of NTM, it is faster and usually gives results that concur with those from the culture. The appropriate combination of diagnostic methods with clinical correlation are necessary.