In the Arctic fjords, the marine mycobiome experiences significant changes under environmental conditions driven by climate change. However, research on the ecological roles and the adaptive mechanisms of marine mycobiome in the Arctic fjord remains insufficiently explored. The present study employed shotgun metagenomics to comprehensively characterize the mycobiome in 24 seawater samples from Kongsfjorden, a High Arctic fjord situated in Svalbard. It revealed the presence of a diverse mycobiome with eight phyla, 34 classes, 71 orders, 152 families, 214 genera, and 293 species. The taxonomic and functional composition of the mycobiome differed significantly among the three layers, i.e., upper layer (depth of 0 m), middle layer (depths of 30–100 m), and lower layer (depths of 150–200 m). Several taxonomic groups (e.g., phylum Ascomycota, class Eurotiomycetes, order Eurotiales, family Aspergillaceae, and genus Aspergillus) and KOs (e.g., K03236/EIF1A, K03306/TC.PIT, K08852/ERN1, and K03119/tauD) were significantly distinct among the three layers. Among the measured environmental parameters, depth, NO₂⁻, and PO₄³⁻ were identified as the key factors influencing the mycobiome composition. Conclusively, our findings revealed that the mycobiome was diverse in the Arctic seawater and significantly impacted by the variability of environmental conditions in the High Arctic fjord. These results will assist future studies in exploring the ecological and adaptive responses towards the changes within the Arctic ecosystems.