The present study describes the chemical composition and the antifungal activity of Vitex agnus-castus L. oil extracted from seeds using Soxhlet apparatus. The Vitex seeds oil contains significant quantity of fixed oil (7.9 ± 0.75 %). Unsaturated fatty acids are the major component of Vitex oil. Oleic and linoleic acids constitute 87% of the fatty acids. While, very low percentage of linolenic acid was found in Vitex seeds oil (<0.4%). -sitosterol and stigmasterol were among the major components, together constituting about 69 % of total sterols. In Vitex seeds oil, cholesterol was found at about 0.1% of the total sterols. Vitex oil has a strong antifungal activity compared with the conventional fungicides. The MIC50, MIC90 and the Minimal Fungicidal Concentration (MFC) were respectively 1.75, 7 and 7 (mg/ml) against all tested Candida species isolated from nosocomial infections in hospitals. Our findings demonstrate that Vitex oil possesses antifungal activity that might be a natural potential source of antifungal used in food, in cosmetics and in pharmaceuticals products.