In recent decades, by increasing immunocompromised patients, disease related to nontuberculous mycobacteria, previously known as environmental opportunistic pathogen, has been raised. In this study, 85 water samples from different sources in Isfahan were evaluated for the presence of NTM. Phenotypic tests were used to identify NTM species. Twenty one out of 85 (24.7%) collected water samples had at least one NTM. Of these, 23.8% (5 isolates) and 14.3 % (3 isolates) were M. furtuitum and M. smegmatis, respectively. Two cases for each isolates (9.5%) were identified as M. chelonae like organisms, M. terrae complex, M. gordonae and M. mucogenicum. One cases for each isolates (4.8%) was determined as M. avium complex, M. phlei, M. xenopi, M. fallax, and M. flavescence. The results showed the incidence of different species of NTM in this geographical region in Iran. Because of increasing immunocompromised disease in communities, and high frequency of NTM in different geographical regions, understanding the NTM distribution in environment would help clinicians to manage proper treatment strategy.