Aims To investigate the antifungal activity of conventional chitosan and chitosan-loaded nanoemulsions against anthracnose caused by Colletotrichum spp. isolated from different tropical fruits. Methods and Results In vitro results illustrated that conventional chitosan onwards 1.5% concentration inhibited radial mycelial growth, conidial germination, sporulation and dry weight of mycelia for Colletotrichum musae (Berk. & Curt) Arx. isolated from banana, Colletotrichum gloeosporioides (Penz.) Penz and Sacc. isolated from papaya and dragon fruits. For further investigations, chitosan-loaded nanoemulsions were prepared, and chitosan at 2.0% concentration with 200 nm droplet size gave the best results in terms of all the in vitro parameters tested for C. musae and at the same concentration with 600 nm droplet size for both the isolates of C. gloeosporioides. However, the results obtained at 2.0% chitosan concentration with different droplet sizes were nonsignificantly different with 1.0 and 1.5% concentrations. Therefore, for in vivo studies, only 1.0% chitosan with different droplet sizes was used. In terms of fungicidal effects and maintaining postharvest quality of banana, papaya and dragon fruits, chitosan at 1.0% concentration with a droplet size of 200 nm in banana and 600 nm in papaya and dragon fruits showed the best results in delaying the onset of anthracnose and maintaining quality of all the fruits for up to 28 days of cold storage. Conclusion Chitosan used in a conventional form showed good results but not as effective as in the form of nanoemulsions. Significance and Impact of the Study The results of this study suggest that instead of applying chitosan in a conventional form, chitosan in the form of nanoemulsions could be more effective as a biofungicide for controlling anthracnose of fresh fruits. Moreover, it could be cost-effective as the amount of chemical used is reduced when applied in the form of nanoemulsions.