The chemical composition of essential oils obtained by hydrodistillation from leaves, branches and female cones of Tunisian Cupressus sempervirens L. was determined by gas chromatography (GC) and CG-mass spectrometry (GC/MS) analysis, 52 compounds were identified; qualitative and quantitative differences between oils were observed. All oils were rich in monoterpene hydrocarbons, and the major constituents were α-pinene (27.5 to 35.8%), α-cedrol (7.7 to 19.3%), δ-3-carene (5.8 to 13.2%) and germacrene D (3.9 to 12.1%). Essential oils of C. sempervirens have shown a significant phytotoxic effect against the germination and seedling growth of four weeds: Sinapis arvensis L., Trifolium campestre Schreb (dicots), Lolium rigidum Gaud and Phalaris canariensis L. (monocots). Tested oils strongly inhibited the germination and seedling growth of all weeds, in a dose dependent manner. The in vitro antifungal activity of the essential oil samples of C. sempervirens were evaluated against 10 cultivated crop fungi, and all samples have shown a significant antifungal activity against all tested fungi and it can be suggested to have the potential to be used as a bio-herbicide and alternatives fungicide. Key words: Cupressus sempervirens, essential oils, bio-herbicidal activity, antifungal potential, weeds, allelopathy.