Phytopathological studies of chitosan nanocomposites are mainly focused on in vitro efficiency, so it is essential to perform a complementary greenhouse assay to find eco-friendly alternatives for plant disease management. In the present study, Cu-chitosan and Zn-chitosan nanocomposites were prepared by reduction of metal precursors in the presence of chitosan in sc CO₂ medium and deposition of organosol on chitosan, respectively. Physicochemical properties of the nanocomposites were characterized by X-ray fluorescence analysis (XRF), Small angles X-ray Scattering (SAXS), X-ray Photoelectron spectroscopy (XPS), and Transmission electron microscopy (TEM). The bimetallic blends (BBs) based on nanoscale Cu(OH)₂ were obtained through simple precipitation and grinding methods. In vitro and in vivo studies of the antifungal activity of Cu-chitosan, Zn-chitosan and BBs at concentrations of 30, 60, and 100 μg ml⁻¹ were conducted against two anastomosis groups of Rhizoctonia solani for control of cotton seedling damping-off. Effect of metal-chitosan nanocomposites at 100 μg ml⁻¹ combined with Cu-tolerant Trichoderma longibrachiatum strains was also evaluated for control of cotton seedling damping-off under greenhouse conditions. The BBs and Cu-chitosan nanocomposite showed the highest antifungal efficacy against both anastomosis groups of R. solani in vitro. These results indicated that BBs, Cu-chitosan nanocomposite, and BBs combined with Trichoderma may suppress cotton seedling disease caused by R. solani in vivo. The evaluation of R. solani in a greenhouse with a Trichoderma strain showed synergistic inhibitory effect with BBs. Light micrographs of mycelia treated with BBs showed the disruption of the hyphal structures. The interaction of the nanocomposites with DNA isolated from the exposed fungal cells, by means of bonding and/or degradation, was also investigated. DNA interaction in terms of binding and degradation for treated DNA with BBs and chitosan nanocomposites was demonstrated. The results showed the absence of DNA amplification by a microsatellite primed PCR.