High-altitude volcanoes, typical examples of extreme environments, are considered of particular interest in biology as a possible source of novel and exclusive microorganisms. We analyzed the crater soil microbial diversity of Tianchi Volcano, northeast China, by combining molecular and morphological analyses of culturable microbes, and metabarcoding based on Illumina sequencing, in order to increase our understanding of high-altitude volcanic microbial community structure. One-hundred and seventeen fungal strains belonging to 51 species and 31 genera of Ascomycota, Basidiomycota and Mucoromycota were isolated. Penicillium, Trichoderma, Cladosporium, Didymella, Alternaria and Fusarium dominated the culturable fungal community. A considerable number of isolated microbes, including filamentous fungi, such as Aureobasidium pullulans and Epicoccum nigrum, yeasts (Leucosporidium creatinivorum), and bacteria (Chryseobacterium lactis and Rhodococcus spp.), typical of high-altitude, cold, and geothermal extreme environments, provided new insights in the ecological characterization of the investigated environment, and may represent a precious source for the isolation of new bioactive compounds. A total of 1254 fungal and 2988 bacterial operational taxonomic units were generated from metabarcoding. Data analyses suggested that the fungal community could be more sensitive to environmental and geographical change compared to the bacterial community, whose network was characterized by more complicated and closer associations.