Biodiversity is threatened particularly in perennial crop cultivation such as fruit trees or grapevines. If established, agroforestry has the potential to increase biodiversity by providing a higher habitat heterogeneity at the example of grapevine (Vitis vinifera L. cv. Riesling) cultivated together with oak or poplar trees for 12 years. Together with the rhizosphere microbiome, the root metabolome was quantified as an indicator of root exudation. Since the root metabolome does not fully align with the exudate metabolome, we are using the root metabolome as a proxy for the exudate metabolome. The results reveal that co-cultivation of grapevine with trees reduces the nutrient availability in the soil and changes the root metabolome of both, grapevine and trees with a more distinct effect of trees on grapevine than vice versa, particularly for oak. Apparently, root-to-root signalling takes place between trees and grapevine. Co-cultivation of grapevine and oak trees also enhanced the alpha diversity of the microbiome. Correlation analysis revealed strong positive correlations between distinct microbial families and metabolites enriched in the roots of Riesling. Thus, microbiome analyses support the view that root-to-root interaction in mixed cultivation of grapevine with trees is mediated by root exudation.