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Objective: The current study is a report of isolation and identification of Mycobacterium setense from a tap running water in a general surgery ward in a hospital in Isfahan, Iran. Materials and Methods: This bacterium was obtained from a hospital water sample according to the cetylpyridinium chloride 0.005% method on Lowenstein–Jensen slant and characterized to the species level as M. setense using results of phenotypic test and 16S rRNA sequencing. Results: This isolate identified to the species level using the conventional and molecular method as M. setense, this bacterium has highest similarity (100%) with those of M. setense CIP109395 16S rRNA gene sequences. Conclusion: This study can be useful and provide important information for learning about the natural habitats of pathogenic species of nontuberculosis mycobacteria. Furthermore, the molecular tests such as 16S rRNA gene sequencing can be applied for reliable and appropriate identification of mycobacterial species.