Ectomycorrhizal (EM) associations facilitate plant nitrogen (N) acquisition, but the contribution of EM associations to tree N nutrition is difficult to ascertain in ecosystems. We studied the abilities of subtropical EM fungi and nutritionally contrasting Eucalyptus species, Eucalyptus grandis W.Hill ex Maiden and Eucalyptus racemosa Cav, to use N sources in axenic and soil cultures, and determined the effect of EM fungi on plant N use and plant ¹⁵N natural abundance (δ¹⁵N). As measured by seedling growth, both species showed little dependence on EM when growing in the N-rich minerotrophic soil from E. grandis rainforest habitat or in axenic culture with inorganic N sources. Both species were heavily dependent on EM associations when growing in the N-poor, organotrophic soil from the E. racemosa wallum habitat or in axenic culture with organic N sources. In axenic culture, EM associations enabled both species to use organic N when supplied with amide-, peptide- or protein-N. Grown axenically with glutamine- or protein-N, δ¹⁵N of almost all seedlings was lower than source N. The δ¹⁵N of all studied organisms was higher than the N source when grown on glutathione. This unexpected ¹⁵N enrichment was perhaps due to preferential uptake of an N moiety more ¹⁵N-enriched than the bulk molecular average. Grown with ammonium-N, the δ¹⁵N of non-EM seedlings was mostly higher than that of source N. In contrast, the δ¹⁵N of EM seedlings was mostly lower than that of source N, except at the lowest ammonium concentration. Discrimination against ¹⁵N was strongest when external ammonium concentration was high. We suggest that ammonium assimilation via EM fungi may be the cause of the often observed distinct foliar δ¹⁵N of EM and non-EM species, rather than use of different N sources by species with different root specialisations. In support of this notion, δ¹⁵N of soil and leaves in the rainforest were similar for E. grandis and co-occurring non-mycorrhizal Proteaceae. In contrast, in wallum forest, E. racemosa leaves and roots were strongly ¹⁵N-depleted relative to wallum soil and Proteaceae leaves. We conclude that foliar δ¹⁵N may be used in conjunction with other ecosystem information as a rapid indicator of plant dependency on EM associations for N acquisition.