In the Arctic ecosystems, fungi are crucial for interactions between soil and plants, the cycling of nutrients, and the transport of carbon. To date, no studies have been conducted to thoroughly examine the mycobiome and its functional role in various habitats of the High Arctic region. The aim was to unravel the mycobiome in the nine habitats (i.e., soil, lichen, vascular plant, moss, freshwater, seawater, marine sediment, dung, and marine alga) in the Ny-Ålesund Region (Svalbard, High Arctic) using a high-throughput sequencing approach. A total of 10,419 ASVs were detected. Among them, 7535 ASVs were assigned to unidentified phyla, while the remaining 2884 ASVs were assigned to 11 phyla, 33 classes, 81 orders, 151 families, 278 genera, and 261 species that were known. The distribution of the mycobiome was driven by habitat specificity, indicating that habitat filtering is a crucial factor influencing the fungal assemblages at a local scale in this High Arctic region. Six growth forms and 19 fungal guilds were found. The ecological guild (e.g., lichenized, ectomycorrhizal) and growth form (e.g., yeast, thallus photosynthetic) varied significantly among various habitats. In addition, the occurrence of 31 fungal species that are considered to be potential pathogens was determined. These results will increase our understanding of fungal diversity and its functional significance in this distinctive High Arctic area and thereby establish the groundwork for prediction about how the mycobiome will alter in various environments as a result of anticipated climate change. Keywords: fungal diversity; high arctic; habitat specificity; functional role.