Hot springs contain many microorganisms, and a great deal of research has been conducted on the bacteria in hot springs but less on the fungi. Microbial diversity research in hot springs will help to further develop and utilize the microbial resources from hot springs. This is the first study to analyze the fungal diversity and ecological functions in Tangchi hot spring (China). In this paper, the fungal ITS sequences in the water samples of Tangchi hot spring were sequenced by Illumina MiSeq high-throughput sequencing technology and bioinformatically analyzed to explore the fungal diversity and ecological functions in the hot spring. The results revealed that the dominant fungal phylum was Ascomycota, followed by Basidiomycota, Chytridiomycota, and Olpidiomycota, and the dominant fungal genera were Rhizophydium, Aureobasidium, Rhodotorula, and Sclerotinia in Tangchi hot spring. The FUNGuild functional analysis results revealed that the dominant guilds in Tangchi hot spring were plant pathogens followed by the undefined saprotroph and the animal endosymbiont–animal pathogen–endophyte–plant pathogen–undefined saprotroph. This study can fill the gap in people’s understanding of fungal resources and offer theoretical support for understanding the fungal diversity and fungal resource development and utilization in Tangchi hot spring.