Background and aims: Incorporation of biochar into the soil sequesters C for millennia, but the concomitant effects on plant rhizodepositions and nutrient (e.g., nitrogen; N) trade-offs via interactions of heterotrophic microbiota, might offset this sequestration. Methods: Ryegrass (Lolium perenne L.) with and without biochar amendment were pulse labelled in a ¹³CO₂ atmosphere and ¹⁵N fertilizer added. Ryegrass and soils were destructively sampled at 16 and 30 days after seedling emergence. Isotope analysis was coupled with MiSeq sequencing of bacterial (16s rRNA) and fungal (ITS) genes to identify the effect of biochar on the associated microbiota involved in ¹³C allocation into soil aggregates and promotion of ¹⁵N uptake by L. perenne. Results: Biochar increased root biomass and ¹⁵N uptake but decreased rhizodeposited-¹³C recovery from large and small macroaggregates (by 12–57% and 57–72%, respectively). These changes in ¹³C flow and ¹⁵N uptake were accompanied by an increase in microbial biomass, and enhanced negative correlations between bacteria and fungi. O2PLS indicated members of seventeen genera that were correlated with soil stabilization of rhizodeposits in soil and plant N-uptake. For instance, Xanthomonadales (Proteobacteria) and RB41 (Acidobacteria), previously reported to be plant growth promoting rhizobacteria, were found to be positively correlated with ¹⁵N uptake by L. perenne. Conclusions: Our research explored the genera associated with biochar-modified ¹⁵N uptake by Lolium perenne and photosynthate ¹³C allocation into soil aggregates. Future research with SIP is required to fully assess microbial turnover, the ubiquity of similar rhizosphere microbiota and their fundamental importance for sequestration in the plant-soil-microbe-biochar systems.