In Mexico, the Ataulfo mango crop faces significant challenges due to anthracnose, a disease caused by the fungus Colletotrichum gloeosporioides. The need to use eco-friendly fungicides is crucial to avoid the use of harmful synthetic chemicals. This study aimed to prepare chitosan nanoparticles through a simple and effective ultrasound-assisted top-down method, with high antifungal efficiency. The nanoparticles were prepared from chitosan (DD = 85%, MW = 553 kDa) and Tween 20 under constant sonication. The formation of the nanoparticles was initially confirmed by Fourier-transform infrared (FTIR) spectroscopy; and their physicochemical properties were subsequently characterized using scanning electron microscopy (SEM) and atomic force microscopy (AFM). The antifungal potential of the chitosan nanoparticles against the phytopathogen Colletotrichum gloeosporioides was evaluated with isolated fungi obtained directly from mango tissues showing anthracnose symptoms in the state of Guerrero, Mexico. The fungus was identified through SEM imaging, showing a regular and smooth conidial layer, with cylindrical shape (r = 2 µm, h = 10 µm). In vitro tests were conducted with three different concentrations of chitosan nanoparticles to assess their inhibitory effects. After seven days of incubation, a maximum inhibition rate of 97% was observed with the 0.5% nanoparticle solution, corresponding to a fungal growth rate of 0.008 cm/h. At this time, the control mycelial growth was 7 cm, while the treated sample reached a radius of 0.55 mm. These results demonstrated the antifungal effect of the nanoparticles on the membrane and cell wall of the fungus, suggesting that their composition could induce a resistance response. The inhibitory effect was also influenced by the particle size (30 nm), as the small size facilitated penetration into fungal cells. Consequently, the parent compound could be formulated and applied as a natural antifungal agent in nanoparticle form to enhance its activity. The method described in this study offers a viable alternative for the preparation of chitosan nanoparticles, by avoiding the use of toxic reagents.