ABSTRACT The immunocompromised host is at increased risk of Mycobacterium tuberculosis complex and nontuberculous mycobacteria infection. Although Mycobacterium tuberculosis complex is a significant mycobacterial pathogen, nontuberculous mycobacteria causes substantial disease in those with suppressed immune responses. Mycobacterial infections can cause significant morbidity and mortality in this patient population, and rapid identification and susceptibility testing of the mycobacterial species is paramount to patient management and outcomes. Mycobacterial diagnostics has undergone some significant advances in the last two decades with immunodiagnostics (interferon gamma release assay), microscopy (light-emitting diode), culture (automated broth-based systems), identification (direct PCR, sequencing and matrix-assisted laser-desorption ionization–time of flight mass spectrometry) and susceptibility testing (molecular detection of drug resistance from direct specimens or positive cultures). Employing the most rapid and sensitive methods in the mycobacterial laboratory will have a tremendous impact on patient care and, in the case of Mycobacterium tuberculosis complex, in the control of tuberculosis.