Rosmarinus officinalis (family Lamiaceae), a spontaneous aromatic plant widespread in Algeria and widely used in folk medicine, is also known as “Eklil”. Our work focuses on the study of the biological activities of the essential oil of R. officinalis. Its chemical composition was studied by GC/MS. Twenty one compounds were identified: The major compounds were: 1.8cineole (32.58%), Sabinene (15.92%), and Camphene (14.41%). The results show that the essential oil of R. officinalis has strong antimicrobial activity against strains tested, either bacterial (Klebsiella pneumoniae, Staphylococcus aureus, and Streptococcus pneumoniae) or fungal (Penicillium expansum and Alternaria alternata). In addition, we evaluated the toxicity induced by the essential oil of R. officinalis in a model of Drosophila melanogaster. Exposure of adult flies to the essential oil via a fumigation method resulted in a mortality-concentration relationship. The result of this study indicated that the values LC25, LC50, and LC90 of the EO of leaves obtained after 24 h were 50.91, 81.32, and 207.42 μL/Lair, respectively. In light of our findings, attention is drawn to the medicinal use of the plant. In addition, there is potential bio-insecticidal activity.