This study aimed to assess crop yield and soil nitrogen removal by trees from a potential nitrogen leaching pool in Mediterranean agroforestry, in a 15N-labeled fertilizer field experiment. We hypothesized that the applied 15N would be taken up by the trees, understorey vegetation strip (UVS), and soil microbial biomass, and that crop yield would be reduced in agroforestry systems. In a two-block agroforestry design, 15N-labeled urea was applied to the crop-soil surface in agroforestry and monocrop plots. We analyzed the 15N, nitrogen, and carbon contents of the aboveground and belowground compartments of the trees, crops, and UVS, as well as that of the soil microbial biomass. No labelled 15N was found in the trees, UVS and microbial biomass. Additionally, agroforestry-crop yielded a -20% lower yield compared to monocrop. The absence of 15N uptake by trees was attributed to the tree age and rooting pattern, which resulted in the lack of tree roots in contact with the labelled fertilizer. The absence of labelled 15N in the microbial biomass indicated a preference for pre-existing organic matter over newly generated 15N from urea mineralization. The capacity of agroforestry systems on crop yield and potentially leachable nitrate may be influenced by the age of trees and prevailing environmental and management practices. Further investigations after several years of tree growth are necessary to better understand the long-term effects of tree age on crop yield and the nutrient retention capacity, including the response pattern of nitrogen-fixing trees.