Many social insect species build nests, which strongly differ from the surrounding environment and are often occupied by specific organismal communities. In temperate forests, red wood ants (e.g. Formica polyctena) are known to create such distinct, highly developed nests, which consist of large, above-ground mounds. Those structures are built primarily out of plant matter collected from the forest litter. Common fungal dwellers of forest litter are representatives of Mucoromycota, engaged in the decomposition process of this substrate. However, data on co-occurrence or interactions between these ants and fungi remains unknown. In order to elucidate these interactions we characterized Mucoromycota communities of Formica polyctena nests and the surrounding forest litter. We sampled four sites, twice in a season and used: a culturomics approach, complemented with DNA barcoding to describe fungal communities; PERMANOVA test and non-metric multidimensional scaling ordinations to compare those communities; and multilevel pattern analysis to indicate taxa associated with the mounds. Our results show that the Mucoromycota community of Formica polyctena’s mound is specific and more stable than the community of the surrounding forest litter. While representatives of Entomortierella lignicola and Absidia cylindrospora clade were found to be associated with the mound environment, representatives of Umbelopsis curvata and Podila verticillata-humilis clade were associated with forest litter, and were rarely present in the mounds. Our findings strongly suggest that the red wood ants’ nest is a specific microenvironment in the temperate forest floor, which is a preferred microhabitat for the mound-associated Mucoromycota, possibly adapted to live in close proximity to ants.