Soil fungi associated with plant roots, notably ectomycorrhizal (EcM) fungi, are central in above-and below-ground interactions in Mediterranean forests. They are a key component in soil nutrient cycling and plant productivity. Yet, major disturbances of Mediterranean forests, particularly in the Southern Mediterranean basin, are observed due to the greater human pressures and climate changes. These disturbances highly impact forest cover, soil properties and consequently the root-associated fungal communities. The implementation of efficient conservation strategies of Mediterranean forests is thus closely tied to our understanding of root-associated fungal biodiversity and environmental rules driving its diversity and structure. In our study, the root-associated fungal community of Q. suber was analyzed using high-throughput sequencing across three major Moroccan cork oak habitats. Significant differences in root-associated fungal community structures of Q. suber were observed among Moroccan cork oak habitats (Maa A mora, Benslimane, Chefchaoun) subjected to different human disturbance levels (high to low disturbances, respectively). The fungal community structure changes correlated with a wide range of soil properties, notably with pH, C: N ratio (P = 0.0002), and available phosphorus levels (P = 0.0001). More than 90 belowground fungal indicators (P < 0.01)-either of a type of habitat and/or a soil property-were revealed. The results shed light on the ecological significance of ubiquitous ectomycorrhiza (Tomentella, Russula, Cenococcum), and putative sclerotia-associated/ericoid mycorrhizal fungal taxa (Cladophialophora, Oidiodendron) in the Moroccan cork oak forest, and their intraspecific variability regarding their response to land use and soil characteristics.