Fungal diversity is threatened by climate change, land-use change, and environmental pollution, and requires urgent conservation action. Construction of the fungal species database is an important prerequisite for the study and conservation of fungal diversity. Recently developed DNA barcoding and metabarcoding technologies can provide accurate, rapid, and highly efficient identification on a large scale, and to a large extent compensate for the defects of traditional identification methods. In this paper, we review the application of metabarcoding in fungal species diversity assessment, the study of mechanisms underlying fungal diversity, and the reconstruction of fungal palaeoecology. We emphasize that the application of metabarcoding technology in fungal diversity studies is still in the primary phase, and greater efforts are needed in the construction of reliable reference databases, the optimization of experimental procedures, and updates of bioinformatics tools. Hence, we suggest enhancing cooperation among fungal taxonomists, ecologists, and computer technicians. They should work together to address problems in fungal diversity studies via metabarcoding, which would provide more sound scientific evidence for fungal diversity conservation on a large scale.