Mycobacteriosis is a serious chronic disease that affects a wide range of marine and fresh water ornamental fish. The present study analyzed the incidence of mycobacteria in 60 apparently healthy fresh water aquarium fish belonging to eight different species as well as 10 pond water samples from Uttar Pradesh, India. In total, thirteen mycobacterial strains of nine species were recovered, out of which, eleven isolates belonging to seven species were obtained from fish and two isolates of different species from pond water. The proportion of mycobacterial incidence was reported 18.33% in apparently healthy fresh water aquarium fish and 20% in pond water. Isolates were identified on the basis of growth characteristics, phenotypic characters and sequence analysis of the 16S rRNA gene as: three isolates of M. abscessus two isolates of M. fortuitum two isolates of M. conceptionense and one isolate of M. parascrofulaceum, M. gordonae, M. bolletii, M. paraffinicum, M. aubagnense, M. arupense each. Mycobacterial incidence in clinically healthy freshwater aquarium fish and natural water sources is awful and the study is relevant because of the mycobacterial diversity related to aquarium fish and its zoonotic importance.