Colletotrichum fungus complex affects several crops and tropical products, which suffer significant losses due to anthracnose. The use of chitosan nanoparticles (CNPs), alone or in combination with bioactive compounds, has been recommend for agronomic applications. However, there is very little information on their phytotoxicity, and there is no information about the effect on microorganisms with low susceptibility to chitosan. This work aims to compare their antifungal effect against isolates of C. fructicola with low susceptibility to chitosan and to study the toxicological effects of CNPs on the germination of lettuce (Lactuca sativa) and radish (Raphanus sativus) seeds. Levels of phytotoxicity of high-to-very high and moderate-to-very high were observed for lettuce and radish seeds, respectively, with greater detrimental effects on the radicle elongation after exposure to CNPs concentrations of 4-5g L-1. For the three C. fructicola isolates, the CNPs did not inhibit the fungal growth; however, the cell viability decreased as the CNPs concentration increased, and a complete inhibition of the viability was found for H4-1 and 003 isolates at a CNPs concentration of 5g L-1. Morphometric alterations characterized by a reduction in the average length of the terminal hyphae, distortion, and a higher number of branches in the hyphae, were observed. To our knowledge, this is the first report where the effect of nanochitosan particles in Colletotrichum fructicola, with experimentally proven low sensitivity to chitosan, was studied.