The information on the contribution of non tuberculous mycobacteria (NTM) to mycobacterial infections in Africa is scarce due to limited laboratory culture for its isolation and identification. One hundred and thirty-seven sputum smear positive patients were recruited into a study on the molecular epidemiology of Mycobacterium tuberculosis in Cross River State. Following sputum culture, 97 pure isolates were obtained and identified using Capilia TB-Neo and further confirmed by the GenoType Mycobacterium CM kit. Of the 97 isolates, 81 (83.5%) isolates were Capilia TB-Neo positive while 16 (16.5%) were Capilia TB-Neo negative. Further confirmation with the GenoType Mycobacterium CM kit revealed that 4 (25%) of the 16 isolates belonged to NTM and included M. fortuitum I, M. fortuitum II/M magaritense, M. abscessus, and M. avium ssp. The remaining 12 (75%) Capilia TB-Neo negative isolates were not members of the genus Mycobacterium despite their AFB appearance. Six (33.3%) of the Capilia TB-Neo negative were from HIV positive tuberculosis patients. All subjects in this study were placed on DOTS shortly after the AFB results were obtained. The implication of isolation of 16.5% nontuberculous isolates further emphasizes the need for culture of sputum specimen especially in HIV positive patients prior to administration of antituberculosis therapy.