Mycobacteriosis or fish tuberculosis a chronic or acute, systemic, granulomatous disease in fish that occurs due to nontuberculous mycobacteria and affect all fish species. Families of Cyprinidae, Anabantidae and Characidae appears to be more susceptible to mycobacteriosis. This survey was was carried out to investigate the mycobacterial infection of ornamental fish of Semnan. 30 pieces of goldfish and 30 pieces of guppy were collected from aquarium shops of Semnan and sampled. These samples contain kidney liver and spleen. Detection of mycobacteria from extracted DNA was done, using PCR assay.  Partial primer was used to identify the rpoB gene. Results showed contamination of 10% of guppies and 46.66% of goldfish. In DNA sequencing some of the bacterial species was recognized as Mycobacterium fortuitum. In conclusion ornamental fish of Semnan are contaminated with nontuberculous mycobacteria. Considering the zoonotic risk of this disease, results of this survey is important to the general hygiene. Investigating the other areas of Semnan province is offered.