We evaluate the performance of the TBcID assay in a panel of 100 acid-fast bacilli cultures. Sixty-four isolates were TBcID positive for Mycobacterium tuberculosis complex (MTBC), whereas 36 gave negative results. These included 28 nontuberculous mycobacteria, one nonmycobacterial isolate, one M. tuberculosis, and six M. bovis BCG strains. This corresponds to a sensitivity of 90.14%, specificity of 100%, and positive and negative predictive values of 100% and 80.55%, respectively. The test is rapid, easy to perform and interpret, and does not require sample preparation or instrumentation. However, a negative result does not exclude the presence of a strain belonging to MTBC, especially when mutations in mpb64 gene are present or some M. bovis BCG strains are isolated. The TBcID showed potential to assist in the identification of MTBC when the implementation and usage of molecular methods are often not possible, principally in resource-limited countries.