Red Sea fish and shellfish (120 samples) representing 10 species were collected and reared in a contaminated earthen pond at National Institute of Oceanography and Fisheries (NIOF), Hurghada to simulate contaminated aquaculture conditions. The mycobiota associated with fish samples and their environment were examined using two isolation media at 28 °C during the period from October 2014 to September 2015. Twenty-six species related to 11 fungal genera were isolated from the examined fish samples on both media. The most common fungal genera on fishes were; Aspergillus, Penicillium and Purpureocillium. From the above genera the most predominant species were: A. flavus, A. fumigatus, A. niger, A. sydowii, A. terreus, A. versicolor, P. aurantiogriseum and Purpureocillium lilacinum. Cladosporium and Exophiala were isolated from some fish and shellfish in the following descending order; E. xenobiotica, C. sphaerospermum, C. antarcticum, C. velox, C. colombiae and E. salmonis. Fish skin and gills yielded relatively higher fungal count than liver. Ten and 14 species belonging to 2 and 8 genera were isolated from earthen pond water and sediment samples, respectively. Fungal isolation showed a correlation between species isolated from water and sediments and those isolated from fish and shellfish especially for Aspergillus species. Otherwise Penicillium donkii, Fusarium solani and Acremonium strictum were recorded only from sediments. All of the 25 isolates tested for enzyme production were positive for lipase, whereas, 16 (64%) isolates exhibited different capabilities of proteolysis.