Few studies have addressed the occurrence of fungi in deep-sea sediments, characterized by elevated hydrostatic pressure, low temperature, and fluctuating nutrient conditions. We evaluated the diversity of fungi at three locations of the Central Indian Basin (CIB) at a depth of ~5,000 m using culture-independent approach. Community DNA isolated from these sediments was amplified using universal and fungal-specific internal transcribed spacers and universal 18S rDNA primer pairs. A total of 39 fungal operational taxonomic units, with 32 distinct fungal taxa were recovered from 768 clones generated from 16 environmental clone libraries. The application of multiple primers enabled the recovery of eight sequences that appeared to be new. The majority of the recovered sequences belonged to diverse phylotypes of Ascomycota and Basidiomycota. Our results suggested the existence of cosmopolitan marine fungi in the sediments of CIB. This study further demonstrated that diversity of fungi varied spatially in the CIB. Individual primer set appeared to amplify different fungal taxa occasionally. This is the first report on culture-independent diversity of fungi from the Indian Ocean.