Ruta chalepensis L. is an aromatic plant belonging to the family Rutaceae, widely distributed in Saudi Arabia. The study focuses on the extraction by steam distillation, phytochemical analysis, GC/MS analysis and antimicrobial activity of essential oil from the leaves of the plant. The results revealed the presence of flavonoids, terpenoids, steroids,and sugars at reasonable concentrations in ethanol, chloroform, ethyl acetate, petroleum ether and n-butanol extract of R. chalepensis leaves. GC/MS analysis identified twenty-eight compounds with the major compounds being 2-Tridecanone (38.88%), 2-Nonanone (29.56%), 2-Acetoxytridecane (18.58%), while 2-Pentadecanol acetate (3.42%), 2-Decanone (1.67%) and 2-Dodecanone (1.25%) were found in small concentrations and the other compounds were found in trace amounts of less than 1% for each. The antimicrobial activity determined by disc diffusion methods showed that the highest activity was observed against S. aureus (inhibition zone of 20.33?0.151 mm), followed by E. coli (inhibition zone of 16.67?0.158 mm), Bacillus cereus and Candida albicans (inhibition zones of 15.67?0.254 mm and 15.33?2.52 mm, respectively) and the lowest activity was against Pseudomonas aeruginosa (inhibition zone of 14.67?1.64 mm). The study, therefore,highlighted on the fact that extracted essential oil could be considered as a potential antimicrobial agent in therapeutic and pharmaceutical applications.