While a recent study showed that significant amounts of the nitrogen (N) requirements of young Eucalyptus trees can be provided by nitrogen-fixing trees (NFTs) in mixed-species plantations through short-term belowground N transfer, the consequences of soil fertility on this facilitation process remain unknown. We assessed the effect of fertilization on the percentage of N derived from transfer (%NDFT) from Acacia mangium trees to Eucalyptus trees in mixed-species plantations. A complete randomized block design with two treatments (fertilized vs unfertilized) and three blocks was set up in mixed-species plantations of A. mangium and Eucalyptus in Brazil, with 50% of each species at 2.5 m × 2.5 m spacing. Collection of litterfall and forest floor made it possible to estimate the annual N release from forest floor decomposition between 46 and 58 months after planting, close to harvest age. 15N-NO3- was injected into the stem of one dominant Acacia tree in each plot, 58 months after planting. The x(15N) values of Acacia and Eucalyptus fine roots sampled within 1.8 m of the labelled A. mangium tree were determined at 7, 14, 30 and 60 days after labelling. The x(15N) values in wood, bark, branch and leaf samples were also determined for the 6 labelled Acacia trees and their two closest Eucalyptus neighbours, just before and 60 days after labelling. The amount of N released from forest floor decomposition was 31% higher in fertilized (F+) than in unfertilized (F-) plots. Sixty days after labelling, the aboveground compartments of Eucalyptus trees were significantly 15N enriched in both treatments. The x(15N) values of Acacia fine roots were higher than background values from 7 days after labelling onwards in F+ and 30 days after labelling in F-. The x(15N) values of Eucalyptus fine roots were higher than background values in both treatments, from 30 days after labelling onwards. Mean %NDFT values were 18.0% in F+ and 33.9% in F- over the first 60 days after labelling, and 22.8% in F+ and 67.7% in F- from 30 to 60 days after labelling. Fertilization decreased short-term transfer belowground of N from Acacia trees to Eucalyptus trees. Our study suggests that belowground facilitation processes providing N from NFTs to Eucalyptus trees in mixed-species plantations are more pronounced in low-fertility soils than in nutrient-supplied stands.