Truffles produced by Tuber species (Ascomycota) represent an additional income in economically marginal areas, especially if cultivating mycorrhized seedlings in dedicated truffle orchards. However, despite the great progress made in cultivation, truffle production has not increased substantially. One of the reasons may be the lack of knowledge on soil as a key component of truffle species habitats. This is the case for Tuber aestivum, whose soil environment has been studied less than those of T. magnatum and T. melanosporum due to the lower economic value of its ascocarps. We did the present study in an experimental truffle orchard submitted to a light tillage before our investigation and characterized by the presence of different host tree species and soil variation in space. We assessed whether truffle production and T. aestivum mycelial DNA in soil varied depending on symbiont tree species, soil type or both by comparing sub-areas characterized by the presence/absence of: i) two landforms; ii) four host tree species; and iii) the truffle brûlé, i.e., the area lacking herbaceous cover when root colonization by T. aestivum occurs.Results show that combined variations in chemical and physical properties determined by soil forming processes can make an area suitable or not for T. aestivum. The establishment of brûlé and higher ascocarp production occurred in the more alkaline and less compact soil type, where the resistance to ascocarp volume enlargement is lower and aeration of the soil surface layer is higher. The tillage carried out in the truffle orchard presumably mimicked the effect of the brûlé on the soil structure, allowing the enlargement of the productive area outside the brûlé itself and showing how the management of a truffle plantation, even if sub-optimal for T. aestivum colonization, can influence its productive capacity. Our results further suggest that even in the best environmental conditions not all host trees will enter in production, while in less suitable soil conditions little or no production should be expected.