In a global context where water will become a scarce resource under temperate latitudes, managing tree plantations with species associations, i.e., forest mixture or agroforestry, could play a major role in optimizing the sustainable use of this resource. Conceptual frameworks in community ecology suggest that, in mixed plantations, environmental resources such as water may be more efficiently used for carbon acquisition and tree growth thanks to niche complementarity among species. To test the hypotheses behind these conceptual frameworks, we estimated water-use efficiency (WUE) for poplar trees grown in a monoculture, in association with alder trees (forest mixture) and in association with clover leys (agroforestry) in an experimental plantation located in northeastern France. Water-use efficiency was estimated (i) at leaf level through gas exchange measurements and analysis of carbon isotope composition, (ii) at wood level through carbon isotope composition and (iii) at tree level with sap flow sensors and growth increment data. We hypothesized that species interactions would increase WUE of poplars in mixtures due to a reduction in competition and/or facilitation effects due to the presence of the N2-fixing species in mixtures. Poplar trees in both mixture types showed higher WUE than those in the monoculture. The differences we found in WUE between the monoculture and the agroforestry treatment were associated to differences in stomatal conductance and light-saturated net CO2 assimilation rate (at the leaf level) and transpiration (at the tree level), while the differences between the monoculture and the forest mixture were more likely due to differences in stomatal conductance at the leaf level and both transpiration and biomass accumulation at the tree level. Moreover, the more WUE was integrated in time (instantaneous gas exchanges < leaf life span < seasonal wood core < whole tree), the more the differences among treatments were marked.