The relatively new Mycobacterium tuberculosis direct test (MTDT) enzymatically amplifies M tuberculosis complex 16s ribosomal RNA. The sensitivity of the test ranges from 75 to 100%, with specificity of 95 to 100%, positive predictive value between 78% and 100%, and negative predictive value between 95% and 100%. Similar test characteristics have been documented in nonrespiratory specimens and in specimens that ultimately grow nontuberculous mycobacterium (NTM). This test allows for rapid identification of M tuberculosis in the smear-positive patient and may greatly improve sensitivity over acid-fast bacilli smear alone. A negative test result with a positive smear suggests infection with NTM or Mycobacterium avium complex. We present a case that illustrates the value of MTDT for analysis of tissue specimens in immunocompromised patients with suspected mycobacterial disease and review the rapidly developing literature about this test. We propose an algorithm using MTDT, acid-fast smear, and mycobacterial culture for the diagnosis and treatment of the immunocompromised patient with suspected mycobacterial infection.