Valued above all others, the white truffle species (Tuber magnatum Picco) is highly dependent on the forest ecosystem and its underground biology. Despite its economic importance, knowledge of its biology and mycorrhizal symbioses remains limited; moreover, natural yields have sharply declined, and cultivation efforts have produced inconsistent results. This study evaluated various forest and mycorrhizal inoculation techniques to promote T. magnatum mycelium development in three Tuscan sites converted to truffle cultivation, using qPCR analysis. Alongside conventional practices like irrigation, mulching, and tillage, an experimental method with a sterile, spore-inoculated soil barrier was tested to improve host root establishment, enhance mycorrhization, and maintain long-term symbiosis for healthy truffle ecosystems. Soil analyses nine months after planting Quercus robur L. seedlings showed significant differences in Tuber magnatum mycelium abundance across sites and treatments. The MA treatment—mycorrhized seedlings combined with a sterile, inoculated substrate and separation diaphragm—produced the highest mycelial levels, underscoring the importance of initial mycorrhization and soil manipulation. These findings provide valuable insights for optimizing forest management and improving truffle cultivation by enhancing mycelial development, a key step toward increasing truffle production.