In this study, we investigated fungal diversity of six Mid-Oceanic Ridge sediment samples collected in the East Pacific and the South Indian Oceans by culture-dependent as well as culture-independent approaches. A total of 97 fungal isolates were cultured, belonging to 7 genera and 10 species, including Penicillium (2 species), Rhodotorula , Meyerozyma , Ophiocordyceps , Vishniacozyma , Aspergillus (3 species) and Phoma . Forty-six operational taxonomic units (OTUs) were identified from 933 clones selected from 12 clone libraries based on the internal transcribed spacers including 5.8S (ITS) and 18S rDNA. Two cultured fungi and 9 OTUs from the clone libraries showed similarities less than 97% with the existing sequences in GenBank, suggesting possible new fungal taxa. Most of the fungi belonged to the Ascomycota, to a lesser extent the Basidiomycota. Five fungal genera including Aspergillus , Rhodotorula , Ophiocordyceps , Phoma and Penicillium were recovered by both culture-dependent and culture-independent approaches. In addition to some common genera such as Aspergillus , Penicillium , Ophiocordyceps , Purpureocillium , Tolypocladium, Rhodotorula and Fusarium , some unique species were each found in the East Pacific and the South Indian Oceans. It is worth mentioning that Ophiocordyceps heteropoda was found at both oceans by culture-dependent and culture-independent approaches, although it was only previously reported from terrestrial habitats.