The ectomycorrhizal fungus Tuber melanosporum fruits in association with Quercus in natural forests of Spain. Some of these stands are managed to keep an open canopy and meet the habitat requirements of the fungus. However, there are few quantitative studies analysing in these forests the relationship between soil environment and T. melanosporum. Eight forest stands which produce T. melanosporum have been monitored for 6 years in order to characterise the below-ground ectomycorrhizal community and to assess its temporal dynamics after experimental canopy opening. The brûlé, the ground where T. melanosporum fruits, shows a distinct ectomycorrhizal community, characterised by lower density of active ectomycorrhizal tips, lower morphotype richness per soil volume, higher abundance of T. melanosporum and lower abundance of Cenococcum geophilum than soil closest to the trunk of the host Quercus ilex. Opening the canopy has not stimulated an increase in T. melanosporum, suggesting that a shift in the soil environment alone will not trigger the formation of new truffières in the short term. The dry climate of these truffières may be a factor as T. melanosporum abundance appears to be sensitive to annual weather conditions.