Although intestinal microbiota play crucial roles in fish digestion and health, little is known about intestinal fungi in fish. This study investigated the intestinal fungal diversity of three coral reef fish (Lates calcarifer,Trachinotus blochii, andLutjanus argentimaculatus) from the South China Sea using a culturable method. A total of 387 isolates were recovered and identified by sequencing their internal transcribed spacer sequences, belonging to 29 known fungal species. The similarity of fungal communities in the intestines of the three fish verified that the fungal colonization might be influenced by their surrounding environments. Furthermore, the fungal communities in different intestines of some fish were significantly different, and the number of yeasts in the hindgut was less than that in fore- and mid-intestines, suggesting that the distribution of fungi in fishes’ intestines may be related to the physiological functions of various intestinal segments. In addition, 51.4% of tested fungal isolates exhibited antimicrobial activity against at least one marine pathogenic microorganism. Notably, isolateAureobasidium pullulansSCAU243 exhibited strong antifungal activity againstAspergillus versicolor, and isolateSchizophyllum communeSCAU255 displayed extensive antimicrobial activity against four marine pathogenic microorganisms. This study contributed to our understanding of intestinal fungi in coral reef fish and further increased the library of fungi available for natural bioactive product screening.