This study investigates the fungal community in a temperate forest using large-scale sequencing of environmental samples. Soil fungal diversity was analyzed by directly isolating small-subunit (SSU) and internal transcribed spacer (ITS) rRNA genes through PCR and high-throughput sequencing. A total of 412 sequence types were identified from 863 fungal ITS sequences, with 112 ITS sequences from other eukaryotic microorganisms. Equal proportions of Basidiomycota and Ascomycota sequences were found in both ITS and SSU libraries, while other fungal phyla were less frequently recovered. Many sequences matched those of mycorrhizal, plant-pathogenic, and saprophytic fungi. Compositional differences were observed among soil samples from different depths, with mycorrhizal species predominating deeper in the soil and saprophytic species in the litter layer. Fungal richness was lowest in the deepest soil horizon. The study shows that fungal richness is comparable to traditional methods but requires extensive sampling. The high rate of new sequence types and the dominance of fungi in the libraries suggest that fungal abundance and diversity in forest soils may be higher than previously thought. Fungi are diverse components of soil microbial communities, functioning as decomposers, mycorrhizal mutualists, and pathogens. Studying these organisms is challenging due to their high diversity, complex substrates, and difficulty in direct observation. Traditional methods, such as culturing and fruiting body collection, have limitations, recovering only a small subset of the community. Sequence-based studies of DNA from environmental samples are revolutionizing microbial diversity understanding. This technique involves PCR amplification, cloning, and sequencing of target DNA regions. It has been used to identify many uncultivated bacterial species and is now being applied to eukaryotic microorganisms. The ITS region, which evolves faster than SSU rRNA, is a critical marker for fungal species identification. The study aimed to determine the feasibility of using sequence-based inventories for soil fungi to quantify species richness and detect correlations between taxonomic composition and environmental variables. It also compared the utility of ITS and SSU rRNA regions for characterizing soil fungi. The results showed that ITS sequences provided finer taxonomic resolution than SSU sequences, with a higher proportion of fungal sequences in ITS libraries. The study highlights the importance of using sequence-based methods to better understand the dynamics of soil fungal communities.This study investigates the fungal community in a temperate forest using large-scale sequencing of environmental samples. Soil fungal diversity was analyzed by directly isolating small-subunit (SSU) and internal transcribed spacer (ITS) rRNA genes through PCR and high-throughput sequencing. A total of 412 sequence types were identified from 863 fungal ITS sequences, with 112 ITS sequences from other eukaryotic microorganisms. Equal proportions of Basidiomycota and Ascomycota sequences were found in both ITS and SSU libraries, while other fungal phyla were less frequently recovered. Many sequences matched those of mycorrhizal, plant-pathogenic, and saprophytic fungi. Compositional differences were observed among soil samples from different depths, with mycorrhizal species predominating deeper in the soil and saprophytic species in the litter layer. Fungal richness was lowest in the deepest soil horizon. The study shows that fungal richness is comparable to traditional methods but requires extensive sampling. The high rate of new sequence types and the dominance of fungi in the libraries suggest that fungal abundance and diversity in forest soils may be higher than previously thought. Fungi are diverse components of soil microbial communities, functioning as decomposers, mycorrhizal mutualists, and pathogens. Studying these organisms is challenging due to their high diversity, complex substrates, and difficulty in direct observation. Traditional methods, such as culturing and fruiting body collection, have limitations, recovering only a small subset of the community. Sequence-based studies of DNA from environmental samples are revolutionizing microbial diversity understanding. This technique involves PCR amplification, cloning, and sequencing of target DNA regions. It has been used to identify many uncultivated bacterial species and is now being applied to eukaryotic microorganisms. The ITS region, which evolves faster than SSU rRNA, is a critical marker for fungal species identification. The study aimed to determine the feasibility of using sequence-based inventories for soil fungi to quantify species richness and detect correlations between taxonomic composition and environmental variables. It also compared the utility of ITS and SSU rRNA regions for characterizing soil fungi. The results showed that ITS sequences provided finer taxonomic resolution than SSU sequences, with a higher proportion of fungal sequences in ITS libraries. The study highlights the importance of using sequence-based methods to better understand the dynamics of soil fungal communities.