Isolation and identification of pathogenic fungi from African Catfish (Clarias gariepinus) eggs and adults in Ethiopian National Fishery and Aquatic Life Research Center hatchery was conducted from October 2015 to May 2016. The aim of this study was to investigate the aquatic fungal flora associated with eggs and brood stock from African catfish in the center. A total of 16 egg samples, 77 swab samples from skin of adult fishes (brood stock) and water samples from 14 incubating containers were investigated. Samples were collected from water, outer body surface of fish as well as from artificially hatched eggs. Isolation and identification of the fungus was done on colonial and microscopic characteristics. In this study, 84.11% samples were positive for fungal growth and fungus isolates belonging to seven different genera were identified. Tricophyton, Saprolegnia, Rhizopus, Penicillium, Mucor, Microsporum and Alternaria were among the dominant isolated fungal genera. Among the isolated genera, Tricophyton was detected from 13.08% of the samples, while Alternaria had the least with 3.74%, unidentified fungi accounts for 14.02% and unidentified yeast were 20.56%. Based on the results, various pathogenic fungi species were identified from fish body, water and hatched eggs, hence are potential causes of mortality and decreased egg hatchability. In the light of this, appropriate egg and water disinfection methods are essential for hatchery management. Fishes in rearing facilities must be given proper health management monitoring to prevent outbreak of fungal disease.