In developing countries where tuberculosis is still a health challenge, the prevalence of nontuberculous mycobacterial diseases is expected to rise as medical conditions that compromise the immune system become more widespread. In the current study, we aimed to determine the presence and diversity of nontuberculous mycobacteria (NTM) causing infections in Iranian patients. Sixty-seven clinical NTM isolates were identified using conventional and molecular methods, including PCR-restriction fragment length polymorphism analysis (PRA) and 16S rRNA sequencing. Out of 67 patients with confirmed mycobacterial infection, 29 had an associated immunosuppressive syndrome, including 9 who were HIV-infected. Forty-nine NTM isolates were identified using PRA, and the remaining 18 isolates were identified using 16S rRNA sequencing. We obtained the following results: Mycobacterium fortuitum, 30 isolates; M. kansasii, 12 isolates; M. gordonae, 8 isolates; M. porcinum, 3 isolates; M. conceptionense, 3 isolates; M. phlei, 2 isolates; and M. austroafricanum, M. avium, M. elephantis, M. intracellulare, M. lentiflavum, M. monacense, M. parascrofulaceum, and M. thermoresistibile, 1 isolate each; and 1 potentially novel mycobacterial species. With regard to the complexity of identification, it is recommended that laboratory diagnosis of NTM diseases be centralized by strengthening or setting up quality national and regional infrastructure.