Mitigation of N2O emissions, a potent greenhouse gas, remain challenging due to knowledge gaps in plant-mediated nitrogen (N) transformation pathways, which limits ability to identify optimal approaches for efficient N utilization. We set up mesocosms with barley, Italian ryegrass, and barley in combination with Italian ryegrass to assess role of cover crop in N2O emission mitigation. Soil emitted N2O was collected simultaneously from the pots with plants at three growth stages: namely, vegetative, canopy expansion, and grain filling. The gas sample N2O contents, N in microbial biomass (MBN), mineral N content, and phospholipid fatty acid (PLFA) analysis in soils were determined at the three growth stages. Cumulatively, highest N2O was emitted from soil under Italian ryegrass (0.056 mg N g−1 soil) followed by barley (0.0051 mg N g−1 soil) and the least under barley and Italian ryegrass combination (0.0014 mg N g−1 soil). The high emissions under Italian ryegrass occurred at vegetative stage due to high reactive N availability. Strong emissions were observed at canopy expansion stage under barley and were linked to access to the large mineral N proportion redistributed to the lower depth as depicted by highest MBN (0.025 mg N g−1 soil) and decreased extractable N (0.0068 mg N g−1 soil). The high emissions under barley correlated with high fungal/bacterial ratio, pointing towards a fungal role in the emissions. The least soil N2O emissions under barley and Italian ryegrass combination were accompanied by elimination of variations induced by the plant growth stages. Absence of 18:2ω6,9 fungal PLFA biomarker under barley and Italian ryegrass combination indicates a potential inhibition and corresponds with reduced N2O emissions. Together, these results broaden our understanding on how plant-soil interactions drives N2O emissions processes and improves our ability to identify optimal plant-based emission mitigation approaches.