Culture collections (CCs) play an important role in the ex situ conservation of biological material and maintaining species and strains, which can be used for scientific and practical purposes. The Komarov Botanical Institute Basidiomycetes Culture Collection (LE-BIN) preserves a large number of original dikaryon strains of various taxonomical and ecological groups of fungi from different geographical regions. Started in the late 1950s for the investigation of Basidiomycetes’ biological activity, today, in Russia, it has become a unique specialized macromycetes collection, preserving 3680 strains from 776 species of fungi. The Collection’s development is aimed at ex situ conservation of fungal diversity, with an emphasis on preserving rare and endangered species, ectomycorrhizal fungi, and strains useful for biotechnology and medicine. The main methods applied in the collection for maintaining and working with cultures are described, and the results are presented. Some problems for the isolation and cultivation of species are discussed. The taxonomical structure and variety of the strains in the collection fund are analyzed, and they show that the taxonomical diversity of fungi in the LE-BIN is commensurable with the largest CCs in the world. The achievements from the ex situ conservation of the diversity of macromycetes and the main results from the screening and investigation of the collection’s strains demonstrate that a number of strains can be prospective producers of enzymes (oxidoreductases and proteases), lipids, and biologically active compounds (terpenoids, phthalides, etc.) for biotechnology and medicine.