Context Fungi represent a large part of soil biodiversity as well as an essential role for tree hydromineral nutrition, survival, and carbon cycling. While their local diversity has proven to be shaped by abiotic and biotic factors related to soil, climate and vegetation, their response to landscape fragmentation is still debated. Objectives In this paper, we focus on ancient forests characterized by the presence of beech at low elevation, a habitat particularly fragmented in South-West France. We aim to assess the effect of past and present forest fragmentation and quantity on soil fungal and functional group diversity. We expect a negative impact of fragmentation and a positive effect of forest quantity on soil fungal diversity, in addition to an influence of local soil factors. Methods Soils from 41 1ha ancient forest plots across South-West France were sampled along gradients of past and present forest fragmentation and quantity, before their fungal diversity was characterized by metabarcoding of environmental DNA. Hill numbers have been computed and applied to all fungi, trophic guilds and growth forms. Results We find that past forest fragmentation negatively impacts on soil fungal diversity and in particular for symbiotrophs, while the present forest quantity has a positive impact. In contrast, the species richness of pathotrophic fungi shows a negative correlation with past forest fragmentation. The diversity of fungal groups with lower dispersal abilities (e.g., corticioid, microfungi) is negatively impacted by past forest fragmentation, while the diversity of wind-dispersed fungi (e.g., agaricoid) is not influenced by any landscape descriptors. Conclusions Our results show the complexity of fungal responses to past and present forest fragmentation and demonstrate the long-lasting effect of past fragmentation as well as the positive impact of recent forest quantity in the landscape on fungal diversity associated with ancient forest soils. Our study also highlights the huge diversity of fungi unraveled by eDNA metabarcoding in this habitat and the potential of such techniques to study the landscape ecology of soil communities.