Hydrophobic deep eutectic nanoemulsions derived from fatty acids and terpenes (referred to as HyDEN) were introduced as potential nano-coating to effectively combat anthracnose during the postharvest preservation of fruits. Anthracnose primarily caused by the Colletotrichum gloeosporioides species is known for causing substantial damage to fruits. Synthetic fungicides have proven to be effective but come with environmental, health, and safety concerns. In this study, hydrophobic deep eutectic solvents (HDESs) were evaluated for their physical properties to identify the most suitable HDES for the formulation of HyDEN. Menthol-Thymol (MT) at 1:1 and 2:1 ratio met the criteria and was chosen for the formulation of HyDEN. HyDEN (MT-N 1:1) displayed a higher zeta potential value and smaller mean droplet diameter and then tested for its antifungal properties against C. gloeosporioides compared with selected antifungal agents. HyDEN (MT-N 1:1) exhibited strong antifungal activity against C. gloeosporioides and was attributed to its potent mode of action, binding affinity, and rapid onset compared to HDES and fungicide (Globus 5.5). This study also investigated how HyDEN (MT-N 1:1) affects mycelial growth, cell membrane permeability, and cellular leakage of C. gloeosporioides, highlighting its superior disruption of cell membranes compared to HDES and Globus 5.5. This research presents an eco-friendly alternative to the use of toxic chemicals and showcases an innovative and sustainable approach to controlling anthracnose in postharvest fruit preservation.