Fungal root endophytes, including the common group of dark septate endophytes (DSEs), represent different taxonomic groups and potentially diverse life strategies. In this study, we investigated two unidentified helotialean lineages found previously in a study of DSE fungi of semiarid grasslands, from several other sites, and collected recently from a pezizalean truffle ascoma and eggs of the cereal cyst nematode Heterodera filipjevi. The taxonomic positions and phylogenetic relationships of 21 isolates with different hosts and geographic origins were studied in detail. Four loci, namely, nuc rDNA ITS1-5.8S-ITS2 (internal transcribed spacer [ITS]), partial 28S nuc rDNA (28S), partial 18S nuc rDNA (18S), and partial RNA polymerase II second-largest subunit (RPB2), were amplified and sequenced for molecular phylogenetic analyses. Analyses of similar ITS sequences from public databases revealed two globally distributed lineages detected in several biomes from different geographic regions. The host interaction of isolates from nematodes was examined using in vitro bioassays, which revealed that the fungi could penetrate nematode cysts and colonize eggs of H. filipjevi, confirming observations from field-collected samples. This is the first report of a DSE, and we are not aware of other helotialean fungal species colonizing the eggs of a plant-parasitic nematode. Neither conidiomata and conidia nor ascomata formation was detected in any of the isolates. Based on molecular phylogenetic analyses, these isolates represent a distinct lineage within the Helotiales in the Hyaloscyphaceae. For this lineage, we propose here the new genus Polyphilus represented by two new species, P. sieberi and P. frankenii.