Nanochitosan (NCH) has previously been shown to possess antifungal activity against Colletotrichum species (C. chrysophilum, C. siamense, and C. fructicola) isolated from infected Mangifera indica leaves and fruit. However, the molecular response of this phytopathogenic fungus to NCH is still unclear. The objective of this work was to study the gene expression profiles of NCH-treated/untreated Colletotrichum species to gain insight into their molecular responses using RNA sequencing (RNA-seq). Samples for RNA-seq were obtained from the mycelia of each Colletotrichum species, which were either left untreated (control) or treated with NCH (2 mg/mL). The transcriptomes from C. chrysophilum, C. siamense, and C. fructicola were obtained by sequencing with the MGISEQ-2000 platform. The results revealed a differential expression of a total of 1787 (C. chrysophilum), 362 (C. siamense), and 3156 (C. fructicola) genes isolated from leaves, and 3976 (C. fructicola) genes isolated from fruit. Functional annotations showed that DEGs associated with the fungal infection process and the NCH response mechanism were mainly involved in cell wall modifications and oxidative stress responses. Our findings suggest that NCH acts by altering the fungal cell wall’s integrity and mitigating oxidative stress. These findings were similar in the three Colletotrichum species, suggesting a conserved mechanism. Further studies are required to support this assumption.