Although the Pleosporaceae is one of the species-richest families in the Pleosporales, research into less-explored substrates can contribute to widening the knowledge of its diversity. In our ongoing survey on culturable ascomycetes from freshwater sediments in Spain, several pleosporacean specimens of taxonomic interest were isolated. Phylogenetic analyses based on five gene markers (ITS, LSU, gapdh, rbp2 and tef1) revealed that these fungi represent so far undescribed lineages, which are proposed as two novel genera in the family, i.e., Neostemphylium typified by Neostemphylium polymorphum sp. nov., and Scleromyces to accommodate Scleromyces submersus sp. nov. Neostemphylium resembles the genera Gibbago and Stemphylium in the production of phaeodictyospores from apically, somewhat swollen and darkened conidiogenous cells, but differs by the presence of a synnanamorph that consists of blastic, cylindrical and brown phragmoconidia growing terminally or laterally on hyphae, and by its ability to produce secondary conidia by a microconidiation cycle. Scleromyces is placed phylogenetically distant to any genera in the family and only produces sclerotium-like structures in vitro. The geographic distribution and ecology of N. polymorphum and Sc. submersus were inferred from metabarcoding data using the GlobalFungi database. The results suggest that N. polymorphum is a globally distributed fungus represented by environmental sequences originating primarily from soil samples collected in Australia, Europe, and the USA, whereas Sc. submersus is a less common species that has only been found associated with one environmental sequence from an Australian soil sample. The phylogenetic analyses of the environmental ITS1 and ITS2 sequences revealed at least four dark taxa that might be related to Neostemphylium and Scleromyces. The phylogeny presented here allows us to resolve the taxonomy of the genus Asteromyces as a member of the Pleosporaceae.