This study aimed to assess the richness and diversity of aquatic hyphomycetes in the Brazilian Cerrado areas located in the extreme western region of Bahia and in the state of Tocantins, where, to date, no studies involving these organisms have been published. Submerged substrates (leaves and twigs) were collected in six bodies of water: the Azuis River (belonging to the Tocantins River basin), recognized as the smallest river in Latin America and the third smallest worldwide; and in the Grande River, Ondas River, João Rodrigues River, Ouro River, and Janeiro River (part of the São Francisco River basin). The substrates underwent the moist chamber method for subsequent taxonomic study and ecological analysis of the taxa found. Twenty-three aquatic hyphomycetes were found, distributed across three classes, 13 orders, and 20 genera, within the phylum Ascomycota. Among these, 19 are facultative aquatic, three are aero-aquatic, and one is ingoldian. The Azuis (10) and Ondas (9) Rivers exhibited the highest richness and diversity of taxa, whereas the Grande River showed the lowest richness. Concerning the substrate, nine taxa were identified on leaves, 10 on twigs, and four on both. Subulispora procurvata Tubaki was the most abundant species throughout the study, and Delortia palmicola Pat. is the first record of this species for Brazil and for the Cerrado biome. The identification of these fungal species in previously unexplored aquatic ecosystems emphasizes the necessity for additional inventory studies of hyphomycetes in these areas to attain a comprehensive understanding of the funga present in these aquatic environments.