Cryoconite holes on glacier surfaces are a source of cold-adapted microorganisms, but little is known about their fungal inhabitants. Here, we provide the first report of distinctive fungal communities in cryoconite holes in the Werenskiold Glacier on Spitsbergen (Svalbard Archipelago, Arctic). Due to a combination of two incubation temperatures (7 °C and 24 ± 0.5 °C) and two media during isolation (PDA, YPG), as well as classical and molecular identification approaches, we were able to identify 23 different fungi (21 species and 2 unassigned species). Most of the fungi cultured from cryoconite sediment were ascomycetous filamentous micromycetes. However, four representatives of macromycetes were also identified (Bjerkandera adusta, Holwaya mucida, Orbiliaceae sp., and Trametes versicolor). Some of the described fungi possess biotechnological potential (Aspergillus pseudoglaucus, A. sydowii, Penicillium expansum, P. velutinum, B. adusta, and T. versicolor), thus, we propose the Arctic region as a source of new strains for industrial applications. In addition, two phytopathogenic representatives were present (P. sumatraense, Botrytis cinerea), as well as one potentially harmful to humans (Cladosporium cladosporioides). To the best of our knowledge, we are the first to report the occurrence of A. pseudoglaucus, C. allicinum, C. ramotenellum, P. sumatraense, P. velutinum, P. cumulodentata, B. adusta, and T. versicolor in polar regions. In all likelihood, two unassigned fungus species (Orbiliaceae and Dothideomycetes spp.) might also be newly described in such environments. Additionally, due to experimenting with 10 sampling sites located at different latitudes, we were able to conclude that the number of fungal spores decreases as one moves down the glacier. Considering the prevalence and endangerment of glacial environments worldwide, such findings suggest their potential as reservoirs of fungal diversity, which should not be overlooked.