Although dark septate endophytes (DSE) represent a worldwide dispersed form group of root-colonizing endophytic fungi, our knowledge on their role in ecosystem functioning is far limited. In this study, we aimed to test if functional diversity exists among DSE fungi representing different lineages of root endophytic fungal community of semiarid sandy grasslands. To address this question and to gain general information on function of DSE fungi, we adopted api-ZYM and BioLog FF assays to study those non-sporulating filamentous fungi and characterized the metabolic activity of 15 different DSE species. Although there were striking differences among the species, all of the substrates tested were utilized by the DSE fungi. When endophytes characteristic to grasses and non-grass host plants were separately considered, we found that the whole substrate repertoire was used by both groups. This might illustrate the complementary functional diversity of the communities root endophytic plant-associated fungi. The broad spectra of substrates utilized by these root endophytes illustrate the functional importance of their diversity, which can play role not only in nutrient mobilization and uptake of plants from with nutrient poor soils, but also in general plant performance and ecosystem functioning. Interspecific functional diversity of dark septate endophytes was revealed by tests of their substrate utilization and enzymatic capability.