The extensive use of agrochemicals for crop protection is increasing their environmental risks. Due to the incredible antimicrobial potential of nanomaterials, research into their potential use in sustainable agriculture as alternatives to chemical fungicides is advancing rapidly. In this study, we evaluated the possible antifungal properties of copper oxide nanocomposite (CH@CuO NPs) coated with chitosan in order to fend off Fusarium wilt diseases in tomato plants caused by F. oxysporum f. sp. lycopersici (FOL) throughout in vitro and in vivo experiments. Here, we demonstrate some of the characteristics of a potential antifungal nanocomposite composed of copper oxide nanoparticles (CuO NPs), firmly immobilized on chitosan nanoparticle (CH) surfaces as dark spots, with an irregular shape and 54.22 nm in size, as indicated by Transmission electron microscope (TEM) analysis. Spectroscopic and microscopic investigations, as well as its antifungal efficacy, verified that the successful synthesis of the CH@CuO NPs at three different concentrations (1, 25, and 50) mg/L against three different wild isolates of the pathogenic Fusarium oxysporum that infect tomatoes was successfully proven to be effective. In vitro comparisons revealed that CH@CuO NPs showed stronger antifungal activity at only 1 mg/L (96.22 ± 1.35) than the classical chemical fungicide “Kocide 2000” at conc. 2.5 g/L (77.34 ± 0.33), for example, in the case of FOL1 isolate. In accordance with the in vivo data, tomato plants can be treated with only 1 mg/L of CH@CuO NPs for up to 75 days, by which time Fusarium wilt disease severity is reduced by 91.5% In contrast, 2.5 g/L of Kocide 2000 is required to reduce disease in tomato plants by about 90%. This research expands our understanding of agro-nanotechnology by outlining the characteristics of a unique, environmentally friendly, and economically viable nanopesticide for long-term plant protection.