This work aims to study the chemical composition and to evaluate the antimicrobial activity, for the first time, of essential oil and non-volatile extracts of Artabotrys velutinus against Klebsiella pneumoniae 818E, Escherichia coli ATCC 25922 and Staphylococcus aureus ATCC 25933, three clinical strains of reference. The chemical analysis of essential oil of Artabotrys velutinus by GC and GC-MS showed that this oil rich in aromatic components (62.6%) and sesquiterpene hydrocarbons (29.9%) contains 30 compounds representing 98.9% of the oil. The major components of the essential oil were benzyl benzoate (61.2%) also called ascabiol with and E-β-caryophyllene (9.1%). The phytochemical screening of leaves powder of Artabotrys velutinus revealed the presence of saponins, catechin tannins, mucilages, flavonoids, alkaloid, anthocyanins, leuco-anthocyanins, reducing compounds, sterols and terpenes. The in vitro antibacterial activity of the extracts by agar diffusion method showed that only the ethanolic extract of the plant was more effective against E. coli with the highest inhibition zone of 13 mm at 100mg/mL and Minimal Inhibitory Concentration equal to 50 mg/mL. However, the activity of ethanolic extract of this plant was less active than those of reference antibiotics chloramphenicol and gentamycin which were very effective against the strains tested. In sum, essential oil of Artabotrys velutinus and its hydroethanolic extract present weakness antimicrobial activity contrary to its ethanolic extract which possesses moderate activity against clinical strains tested. This study suggests the used of ethanolic extract of Artabotrys velutinus in combination with others active extracts to fight against E. coli.