Aims <p>Nitrogen losses and metabolic imbalance limit nitrogen use efficiency in wheat under conventional urea fertilization. This study examined whether nitrogen nano-fertilizers (N-NFs) improve nitrogen assimilation, reduce nitrogen leaching, and stabilize soil biochemical responses compared with urea.</p> Methods <p>A field experiment was performed using 150 genetically diverse wheat cultivars grown under uniform agronomic management. Urea and N-NFs were applied at three nitrogen concentrations (150, 200, and 250 mg L⁻¹) in a factorial design. Agronomic traits, nitrogen accumulation and leaching, nitrogen-assimilatory enzymes, oxidative stress indicators, antioxidant enzymes, and soil biochemical parameters were analyzed.</p> Results <p>Across nitrogen concentrations, N-NFs increased grain yield by 10–18% relative to urea and were associated with 60–80% higher activities of glutamine synthetase, glutamate synthase, and nitrate reductase, indicating greater nitrogen assimilation capacity. This enzymatic coordination coincided with 40–50% lower leaf ammonia and reduced hydrogen peroxide and lipid peroxidation, reflecting improved redox regulation at the plant level. In soil, N-NFs maintained higher dehydrogenase activity, moderated urease-driven nitrogen transformation, and stabilized electrical conductivity compared with urea. Nitrogen leaching increased markedly under urea at higher concentrations, whereas N-NFs substantially restricted leachate losses, indicating improved nitrogen retention within the plant–soil continuum.</p> Conclusions <p>Nitrogen nano-fertilizers improved nitrogen use efficiency at the plant and soil levels while limiting nitrogen losses to the environment. The novelty of this work is the field-scale evaluation across a 150-cultivar wheat diversity panel combined with integrated measurement of plant metabolism, soil enzymatic responses, and leaching within one factorial framework, enabling assessment of response stability across broad genetic backgrounds.</p> Graphical Abstract <p></p>