The present study aimed to isolate and identify mycobacteria from Nile tilapia and fresh water as well as ornamental fish and aquarium water in Sohag Assuit and Qena Governorates. Conventional and recent methods (MB-Redox system) were used for isolation and detection of mycobacteria with comparison between them regarding to the rate of isolation time for growth and growth character of colonies. Three isolates were isolated from fresh water and identified as unclassified atypical mycobacteria and four isolates were recovered from aquarium water and identified as M. fortuitum and M. marinum. Eleven isolates were isolated from goldfish and identified as M. marinum, M. fortuitum and unclassified atypical mycobacteria. The recovery rate of MB-Redox system for mycobacteria was 12%, while in case of L-J medium was 10.66%. The mean time for detection of M. marinum was 3-7 days and 7-10 days for MBRedox system and Lowenstein – Jensen medium, respectively. The mean time for detection of M. fortuitum was 1-3 days for MB-Redox system, while in case of L-J medium was 3-6 days. The mean time for detection of other unidentified atypical mycobacteria was 3-8 days for MB-Redox system, while in case of L-J medium was 3-14 days. The detection of mycobacterial growth on MB-Redox system at different times revealed that the color of MB- Redox tubes appear yellow at time of inoculation changed to pink color after 3 days. The colonies appear as red particles on the surface. After 15 days, the color of MB-Redox medium changed to yellow with red particles on the surface.