The purpose of this investigation was to assess the usefulness of mycobacteremia detection in human immunodeficiency virus (HIV) patients with suspected tuberculosis. The study included 47 patients with suspected tuberculosis and confirmed HIV infection. A first blood sample was incubated in a BACTEC 9050 MB system, while white blood cells isolation was performed on a second blood specimen before incubation in a BACTEC MGIT 960 system. The third specimen was taken from the affected organs of each patient according to their clinical profile. Twelve (25.5%) patients were positive for mycobacterial infection identified by any of the methods used. Ten (21.2%) were positive for Mycobacterium tuberculosis and 2 (4.3%) for M. avium. Six patients were diagnosed by the culture of specimen from affected organs only, whilst three other patients were positive exclusively for blood cultures. Three additional patients were diagnosed by both methods. Four patients with negative cultures were ultimately diagnosed with tuberculosis by measuring the adenosine deaminase levels. Mycobacteremia detection can be used to increase the sensitivity of the diagnosis of tuberculosis and other mycobacteria in patients with HIV. However, it cannot be used as the sole diagnostic method. Clinical specimen cultures do not provide 100% diagnostic accuracy and it is, therefore, critical to further improve the mycobacteria detection sensitivity.