Purpose: In agroforestry systems, the root distributions and properties of the annual and perennial vegetation are poorly known, although they are recognized for fostering soil carbon (C) stocks through annual root-derived C inputs and by altering microbial activity. This study aimed to evaluate the potential contribution of roots from the understory vegetation strip (UVS) and the crop to top- and subsoil C stocks (0–100 cm) to a 3-year-old agroforestry system of the alley-cropping type. Methods: Root biomass, chemical composition, functional traits and anatomical structure were assessed in parallel to a characterization of soil physicochemical and microbiological properties, including enzymatic activity, at different depths and locations perpendicular to the tree line. Results: The root biomass and properties were heterogeneous in the young alley-cropping system due to the presence of different plant communities and the heterogeneity of the soil mineral N content according to the location perpendicular to the tree line. The soil microbiological properties and organic C stocks did not vary horizontally at this stage of agroforestry but should be monitored through multiple-time samples to confirm a differentiation in subsequent years suggested by the tight link between root stoichiometry and microbial extracellular enzymatic activities that we found. Conclusions: Altogether, our results suggested that increasing the root biomass in topsoil in agroforestry systems positively contributes to increasing soil organic C stocks, but in deeper soil layers, an increase in litter inputs with a high C:N ratio might accentuate microbial N limitations and limit soil C storage.