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The European chestnut tree (Castanea sativa Mill.) is widely cultivated throughout the world’s temperate regions. In the Mediterranean region, it has a significant economic role mainly because of the high quality of its edible nuts. The Oomycete Phytophthora cinnamomi is one of the most severe pathogens affecting European chestnuts, causing ink disease and significant losses in production. Ginkgobilobin-2 (Gnk2) in Ginkgo biloba is a secreted protein with a plant-specific cysteine-rich motif that functions as a lectin, and its carbohydrate-binding properties are closely related to its antifungal activity. The binding of lectins to mannose residues of the cell wall of Phytophthora species may disturb and disrup the cell wall structure. This work determined that the amino acid sequence has a signal peptide that directs the final protein peptide to the apoplast. The Cast_Gnk2-like expression was performed and optimized, and different in vitro antagonism tests were done against P. cinnamomi using different purified protein concentrations. As a result of one of these assays, Cast_Gnk2-like significantly reduced the mycelia growth of P. cinnamomi in liquid medium as shown by the mycelia weight (g) in control treatments was 377% higher than in the treatments. These insights reveal the potential of Cast_Gnk2-like for agricultural uses and biotechnological developments for the pathosystem chestnut/P. cinnamomi.