A rapid and correct diagnosis of mycobacterial infections is important for effective patient treatment. Semi-nested-PCR with Fl-16 SOL, 16SOR and 16SNSR primers based on the 16S rRNA gene, under optimized conditions, can detect 499 bp amplified products from all tested mycobacteria. The assay could detect as little as 100 fg of mycobacterial DNA except for rapid growing mycobacteria, whose detection limits ranged from 1 ng to 10 pg. The specificities of the capture probes were assessed with 96 mycobacterial strains (22 species) and 33 nonmycobacterial strains (30 species). The specificities of pAll1, pTbc1 and pMar1 were 94%, 93% and 82%, respectively, and that of pAvi1, pInt1, pChe1 and pFor1 were 100%. The pTbc1 and pAvi1 were tested with DNA from 108 CSF samples, and the sensitivity and specificity of the detection method were 56% and 84% compared to culture and patient histories. The assay should be used for rapid detection and concurrent identification of slow growing mycobacteria without parallel conventional culture verification.