In order to advance our understanding of the microbiology of the quickly disappearing Arctic glaciers, sampling campaigns in two regions considered as the “hot spots” of polar microbiological research, Svalbard and Greenland, were performed. Samples of supra- and subglacial ice, cryoconite, snow, glacial meltwater, and supraglacial water were analysed for microbial diversity, with a combination of culture dependent and independent methods, and for microbial abundance. Cultivation allowed the isolation of around 500 pure cultures of fungi, belonging to 77 species (62 genera), and more than 200 bacterial cultures belonging to 27 species (23 genera). Most frequent cultivated bacterial genera, common to both Greenland and Svalbard samples, were Cryobacterium, Massilia, Pseudomonas, and Sphingomonas. For fungi, the common genera were the psychrophilic basidiomycetous yeasts Mrakia, Phenoliferia, Callizyma, and the psychrotolerant filamentous Penicillium and Cladosporium. Callizyma is a novel genus isolated from Greenland and Svalbard, comprising two novel oligotrophic and psychrophilic species Callizyma psychrophila and Callizyma arctica. Amplicon sequencing of taxonomic markers uncovered a larger fungal diversity of glacial habitats compared to culturomics, and included some unexpected findings, such as the presence of the dermatophytic fungus Malassezia restricta and the extremely halotolerant black yeast Hortaea werneckii. Additionally, the sequencing revealed the presence of early-lineages fungi (Chytridiomycota and Rozellomycota) in all the collected samples. In both, Svalbard subglacial and Greenland Ice Sheet supraglacial habitats, fungal diversity was mainly composed of Basidiomycota (the majority belonging to the Microbotryomycetes class). The most common bacterial phyla recovered with NGS techniques were Proteobacteria, Cyanobacteria, Chroloflexi, Armatimonadetes, Actinobacteria, and Bacteroidetes. Screening for the expression of phenotypes commonly associated with human pathogenicity in environmental Arctic fungi and bacteria, highlighted the presence of thermotolerant and hemolytic strains, which were often also resistant to antifungals or antibiotics. Interactions between selected fungal species and glacier algae blooming on the surface ice of the Greenland Ice Sheet, contributing to its faster melting, indicated that fungi have a beneficial rather than detrimental impact at the beginning of the melting season. Their behaviour possibly switch to saprophytic later in the season when glacier algae start deteriorating. The same fungal species produced antimicrobial compounds active against tested clinically relevant and environmental bacterial species, pointing to their ability to modulate the growth of other microorganisms.