<p>Anthracnose, caused by <i>Colletotrichum</i> fungal species, is among the most prevalent and severe both pre- and postharvest diseases affecting various fruits, including olive, citrus, and avocado. In an era where chemical control is being challenged by fungicide resistance development and strict EU legislation, causing the withdrawal of many established active ingredients used for disease management, metallic nanoparticles (MNPs) represent promising, eco-friendly fungicide-alternatives for controlling pathogens. In this study, the effectiveness and potential fungitoxicity mechanisms of copper nanoparticles (Cu-NPs) against <i>Colletotrichum gloeosporioides</i> strains isolated from orange and avocado fruit were investigated both in vitro and in vivo. Cu-NPs outperformed their ionic counterpart Cu(OH)₂—used as a reference fungicide- in reducing mycelial growth and disease symptoms. Pearson correlation analysis revealed a positive cross-resistance between Cu-NPs and the demethylase inhibitor (DMI) fungicide difenoconazole. A significant antagonism between Cu-NPs and detoxification inhibitors diethyl malate, piperonyl butoxide as well as DMI fungicides difenoconazole, prochloraz, and prothioconazole used against the pathogen was observed. Furthermore, an induced C-14 alpha-demethylase gene (<i>CgCyp51</i>) overexpression was found, at least partly, to account for the observed superior fungitoxic action of Cu-NPs against <i>C. gloeosporioides</i>. Overall, Cu-NPs demonstrate exceptional potential as alternative fungicides against <i>C. gloeosporioides</i>, outperforming conventional treatments through multiple mechanisms. A key mode of action involving disrupting ergosterol biosynthesis, as evidenced by the significant upregulation of the <i>CgCyp51</i> gene in response to Cu-NP exposure was identified for the first time.</p>