The composition of fungal and bacterial communities in three polythermal glaciers and associated aquatic environments in Kongsfjorden, Svalbard was analysed using a combination of cultivation and amplicon sequencing. 109 fungal strains belonging to 30 mostly basidiomycetous species were isolated from glacial samples with counts up to 103 CFU/100 ml. Glaciozyma-related taxon and Phenoliferia psychrophenolica were the dominant species. Unexpectedly, amplicon sequencing uncovered sequences of Chytridiomycota in all samples and Rozellomycota in sea water, lake water, and tap water. Sequences of Malassezia restricta and of the extremely halotolerant Hortaea werneckii were also found in subglacial habitats for the first time. Overall, the fungal communities within a glacier and among glaciers were diverse and spatially heterogenous. Contrary to this, there was a large overlap between the bacterial communities of different glaciers, with Flavobacterium sp. being the most frequently isolated. In amplicon sequencing Actinobacteria and Proteobacteria sequences were the most abundant.