Mycobacterium isolates were retrospectively identified, antibiotics susceptibility test results and basic clinical data were analyzed for the 715, excepted 308 in 1,023 specimens, from a mycobacterial laboratory at a tertiary care hospital from September 2002 to December 2008. Their male to female ratio was 1.12 to 1 (379 male, 336 female). The median age of study population was 47 years (range from 10 to 93 years). Distribution of Mycobacterium species was 90.1% of total were isolates Mycobacterium tuberculosis, and 9.9% of the total non-tuberculosis Mycobacterium isolated, and Among nontuberculosis Mycobacterium isolates, 60.6% were Mycobacterium avium complex, 14.1% were isolates Mycobacterium abscessus, and 12.7% were isolates Mycobacterium intracellulare. Among 526 Mycobacterium tuberculosis isolates, 81.7% isolates were susceptible to first line antibiotics, 18.3% were resistant to one or more antibiotics. Non-tuberculosis Mycobacterium isolates, all were resistant to two or more antibiotics. Multi-antibiotic resistant tuberculosis rate was show 10.2% of total specimens. Isolated Mycobacterium species, 19.2% were multi-antibiotic resistant tuberculosis, and the rate of nontuberculosis Mycobacterium resistant to isoniazid and rifampin was very highly 84.5%. Thus among acid fast bacilli culture positive cases, Mycobacterium tuberculosis and non-tuberculosis Mycobacterium were must exactly identification and antibiotic sensitivity test. It was considered to help to select of the antibiotic in preventive medicine.