Background and Aims <p>Improving agricultural tolerance to climate change is crucial for food security. We investigated whether combining wheat genotypes with contrasting root architecture enhances plant performance under varying conditions. Specifically, we examined how these genotype mixtures affect nitrogen uptake, carbon release and root-microbe interactions compared to single-genotype plantings.</p> Methods <p>We exposed monocultures and a mixture of shallow- and deep-rooting spring wheat (<i>Triticum aestivum</i> L.) genotypes separately to well-watered and water-deficit conditions in a column experiment. We determined plant and microbial biomass, major microbial groups, and β-glucosidase activity using soil zymography. Additionally, we followed carbon and nitrogen fluxes in the plant-soil-microorganism system by ¹³CO₂ labelling of the atmosphere and ¹⁵N injection into top- and subsoil.</p> Results <p>Combining wheat genotypes with contrasting root phenotypes influenced microbial activity and nutrient uptake depending on water availability. Under well-watered conditions, the mixture performed similarly to the respective monocultures. However, under water-deficit conditions, it exhibited complementary nutrient acquisition strategies where the deep-rooting genotype accessed deeper soil layers, while the shallow-rooting genotype relied more on topsoil nitrogen. This was accompanied by a reduced release of plant-derived carbon into the soil, resulting in lower microbial abundance and reduced β-glucosidase activity compared to monocultures.</p> Conclusion <p>Our results show that plants grown in a mixture performed similarly to monocultures under well-watered conditions while acquiring nutrients more efficiently under water-deficit conditions. This highlights the potential suitability of combining genotypes with contrasting root phenotypes under climate change. However, yield effects remained untested due to experimental constraints, warranting further investigation under field conditions.</p>