Despite that the applicability of Cu-based engineered nanoparticles (ENPs) as an antibacterial and antifungal agent for plant protection has been studied widely, little is known about their role in the improvement of crop yield and quality. Here, a full life study was performed to investigate the nutritional quality and bioactivity of barley grains under foliar application of nano-/microparticulate (nano-Cu, nano-CuO, micro-Cu) and ionic Cu compounds (CuSO4, CuEDTA). Hordeum vulgaris L. plants were sprayed with Cu compounds at 500 mg/L during the end of tillering and the beginning of heading. Yield, mineral composition, protein and dietary content, antioxidant (phenolic, anthocyanin, flavonoid, tannin, flavanol) content and antioxidant capacity of barley grain were evaluated. Grain yield was unaffected by all treatments. Only nano-Cu and ionic compounds enhanced Cu accumulation in grain: 2-fold increase was observed compared to the control (2.6 µg/kg). Nano-Cu also increased the dietary fiber content by 19.9 %, while no impact of the other treatments was determined. The content of phenolic compounds, the main group of antioxidants, remained unchanged after Cu supply. In general, for all Cu treatment, antiradical and reducing abilities were decreased or were at the similar level in relation to the control. On the other hand, chelating power in grain extracts was 2–4 times higher under nano-Cu/nano-CuO/micro-Cu than in the untreated sample, while the ionic compounds had no impact on the chelating indicator. Our results demonstrated that more favorable effects were triggered by nano-Cu than CuSO4 or CuEDTA on the tested indicators of barley grain, despite that both compounds resulted in similar superior Cu acquisition. It suggests that nano-Cu may be considered as an alternative agent to be used as economic and traditional fertilizers.