Background <p>Chitosan has been reported to induce host defense responses against plant viruses, while copper oxide nanoparticles can directly inhibit viral infection. However, the combined effect of chitosan-loaded copper oxide (CS-CuO) nanocomposites, particularly across different nanosizes, remains unexplored against Zucchini yellow mosaic virus (ZYMV). Therefore, this study investigates the antiviral potential of CS-CuO nanocomposites of varying mean particle sizes (70.33, 30.20, and 17.18 nm) to determine whether size-dependent enhancement of induced resistance occurs in squash plants.</p> Methods <p>Three CS-CuO nanocomposites with mean particle sizes of 70.33 nm (CS-CuO₇₀), 30.20 nm (CS-CuO₃₀), and 17.18 nm (CS-CuO₁₇) were chemically synthesized and characterized using transmission electron microscopy and dynamic light scattering (zeta potential analysis). Naturally infected squash samples were collected from open fields in the Beni‑Suef Governorate, Egypt (during the 2025 growing season). ZYMV was molecularly detected by RT‑PCR targeting the P1 gene (primers ZY140F/ZY1084R; annealing 64 °C; 35 cycles), yielding a ~ 944 bp amplicon. The amplicon was sequenced, subjected to phylogenetic analysis, and the obtained sequence was deposited in GenBank under accession number PX572932. Under greenhouse conditions, ZYMV‑inoculated squash plants were foliar‑treated 48 h post‑inoculation with each nanocomposite at 50, 100, and 250 mg/L. Untreated healthy and infected plants served as controls, and all treatments were arranged in a completely randomized block design with five biological replicates per treatment.</p> Results <p>ZYMV infection markedly increased oxidative damage, evidenced by elevated malondialdehyde levels, high disease incidence, and severe symptom development. Additionally, viral stress caused a modest increase in osmoprotectants as well as enzymatic (e.g., APX, SOD, CAT, PPO, and GR) and non-enzymatic antioxidants, but severely suppressed photosynthetic pigments and growth parameters. Foliar application of all CS-CuO nanocomposites significantly (<i>p</i> ≤ 0.05) mitigated ZYMV-induced damage by reducing lipid peroxidation, enhancing photosynthetic pigments, and further strengthening antioxidant capacity. These coordinated responses collectively reduced disease incidence and severity while improving plant growth. All reported treatment effects were statistically significant (<i>p</i> ≤ 0.05) according to two-way analysis of variance. Among treatments, CS-CuO₁₇ at 250 mg/L was the most effective, reducing disease incidence and severity by 89.9% and 91.8%, respectively, and markedly enhancing leaf number, shoot and root length, and plant dry weight relative to the untreated infected controls.</p> Conclusions <p>Overall, CS–CuO nanocomposites, particularly the smallest formulation, effectively enhanced squash growth and activated defense responses against ZYMV. These findings highlight CS-CuO nanocomposites as promising, eco-friendly antiviral agents and a novel approach for the sustainable management of ZYMV in cucurbit production systems.</p>