Tuber magnatum Pico, the Italian white truffle, commands the highest prices of any truffle. Despite its commercial value, it is the only edible European truffle which has not yet been successfully cultivated. Because of this, it is essential to safeguard natural truffières and to identify cultural practices to maximize their productivity. Soil tillage is successfully and extensively used in black truffle cultivation to enhance productivity, but its effects are not known on the development of T. magnatum. A recently developed real-time PCR assay was applied to evaluate the effects of tillage (10-15 cm depth) on T. magnatum mycelium in two different natural truffle grounds located in Tuscany and Emilia-Romagna. Tillage effects on bulk density, ectomycorrhizal fungal communities, and ascoma production were also assessed. Tilling significantly increased the quantity of T. magnatum mycelium which seemed to be related to an increase in soil porosity by up to 34%, and the diversity of ectomycorrhizal fungal communities. On the contrary, no significant effects were found on ascoma production. The results highlight that real-time PCR is the most reliable method for evaluating the effects of cultural practices on the development of T. magnatum in soil avoiding long-term studies on fruiting body production.