Fungal contamination is a detrimental factor affecting sawdust media-based shiitake cultivation in greenhouses. During fungal monitoring of greenhouses used for shiitake cultivation, eight fungal species were isolated and identified from indoor air and mushroom flies collected in the greenhouses. The current study reported five species as new in Korea, viz. Ascochyta hordei, Discosia artocreas, Mucor nidicola, Perenniporia medulla-panis, and Pseudozyma prolifica, and confirmed two species, Penicillium charlesii and Penicillium brevicompactum, which were previously recorded in Korea without molecular taxonomic validation. The morphological characteristics and phylogenetic relationships based on nucleotide sequences of the internal transcribed spacer rDNA region or calmodulin gene were described for all identified species.