[Objective] To reveal the diversity and antimicrobial activity of culturable fungi from the deep-sea sediments in 4 different sites in South China Sea.[Methods] We used 4 different methods and 8 different media to study the culturable fungal diversity in 12 deep-sea sediments samples. We identified fungal isolates by colonial morphology observation and ITS-based phylogenetic analysis. We used disc diffusion and growth rate methods to test antibacterial and antifungal activities.[Results] A total of 125 fungi were isolated from the sediments. Based on the ITS sequence analysis and morphological characteristics, the isolates were grouped to 18 fungal species and 12 genera within 2 phyla. Most fungi belong to Ascomycota and only 2 isolates belong to Basidiomycota. The culturable fungal diversity was different among the 4 locations. Most of the fungal isolates exhibited antimicrobial activity. The genera Alternaria, Penicillium and Stemphylium displayed antibacterial activity against more than one indicator. Particularly, Alternaria tenuissima DN09, Alternaria alternata DN14 and Penicillium chrysogenum DN16 displayed activity against both Gram-positive and Gram-negative bacteria.[Conclusion] Higher diversity and antimicrobial activity of the culturable fungi isolated from deep-sea sediments in South China Sea provide the potential application of these fungi.