Alley cropping agroforestry - whereby tree rows are integrated in crop plots - is considered as a lever for the agroecological transition. Its benefit for enhancing soil functioning is rarely studied. We studied soil health in a 25 years temperate agroforestry plot cultivated with barley (Hordeum vulgare) according to two factors: i. the position to the tree row; and ii. the tree species. Soil health was assessed in three positions (in the tree row; in the crop alley next to the tree row and at 6.5 m from the tree row), for three contrasted tree species (Acer monspessulanum, Fraxinus sp., Pyrus communis) using two integrative methods based on soil biological activity (Biofunctool (R), MicroRespTM). The position factor explained soil health differences the best: mean indexes were found 1.6 times higher in the tree row than in both positions in the crop alley, especially the structure maintenance function was impacted (indexes in the tree row = 0.21 to 0.26; indexes in the crop alley = 0.11 to 0.17). Tree species had less impact on soil health and impacted only carbon dynamics and microbial catabolic profiles. Our study invites to consider spatial organization and tree species to optimize soil ecosystem services in agroforestry systems.