Xylem sap exudes from grapevine branches and ensures survival during the dormant period. However, the extensive analysis on the biological and functional characterization of xylem sap has not yet been carried out. Therefore, to comprehend the critical role and potential usage of the xylem sap, we identified and quantified the panel of active ingredients, such as protein, amino acids, carbohydrates, saponins, flavonoids, phenolic compounds, alkaloids, and polyphenols. Results indicates that bleeding sap is slightly acidic, with the accumulation of oxalic acid, tartaric acid, malic acid, lactic acid, and citric acid. Identification and quantification of sugars depicted that xylem sap is mainly rich in fructose, glucose, and lactose. Moreover, distinct amino acids (16), hormones (9), and mineral elements are identified in the bleeding sap. These bioactive compounds are vital for vine growth and development. In addition, xylem sap also has the growth-promoting ability and can be used as a tissue culturing medium for plants. Finally, functional studies suggested that xylem sap possesses antifungal effects that can be considered as a bacteriostatic agent against Botrytis cinerea. Overall, these findings suggested that xylem sap possesses the panel of antioxidants, protein, and growth-promoting and antifungal properties.