The rapid diagnosis of mycobacterial infections is essential to implement the adequate antimicrobial therapy. To verify BacT/ALERT 3D system results by PCR and evaluate its performance against Löwenstein Jensen medium as regards the recovery rate, the time to detection, and contamination rate. Sixty sputum specimens were inoculated in BacT/ALERT MP bottles and on LJ slants. Ziehl-Neelsen [ZN], subculture and PCR was done to confirm the positive signal MP bottles. Thirty two [53.3%] mycobacterial isolates [31 M. tuberculosis and one non-tuberculous mycobacterium] were detected. The recovery rate of 32 mycobacterial isolates for the BacT/ALERT 3D system was 100% and that of LJ medium was 71.9%. Of the 32 signal positive/PCR positive bottles, 27 [84.4%] bottles were ZN smear positive [from the bottle] and 5 [15.6%] were ZN negative. The mean times to detection of mycobacteria by BacT/ALERT 3D system and LJ medium were 14.2 and 24.3 days, respectively, while overall contamination rates were 6.7% and 8.3%, respectively. Sensitivity and time to detection were significantly better with BacT/ALERT 3D system than with solid LJ medium. The PCR assay allows the fast and exact identification of Mycobacterium tuberculosis directly from positive liquid medium. LJ culture still plays an important role in isolation of mycobacteria from clinical samples, it provide visible colonies that allow identification and susceptibility testing of the isolate. Signal positive/ZN negative bottles should be confirmed by other methods [PCR and/or subculture].