We experienced a case of laboratory cross-contamination of Mycobacterium tuberculosis on the broth based culture system. These false-positive cultures were confirmed by analysis of DNA fingerprinting, RFLP method, which showed the same pattern in three specimens with that of the first manipulated specimen in our laboratory on that day, out of 7 specimens examed. We found possible several process causing cross-contamination where mixture of the foreign body could occur in buffer or NALC-NaOH. False-positive cultures for Mycobacterium tuberculosis may lead to unnecessary, potentially toxic, costly treatment, and changing the treatment strategy. So we must critically interpret a single positive culture, especially by liquid media.