In this study, the chemical composition and antimicrobial activity of the essential oil (EO) from aerial parts of Rhetinolepis lonadioides collected from southwestern Algeria were assessed. The EO was obtained by hydrodistillation and analyzed by GC/MS. In total, thirty-two compounds were identified representing 80.00% of the total of the EO, withβ-Pinene (41.85%) being the major monoterpenes. Phenanthroquinone (10.39%) and Ethanone, 1-[1,1'-biphenyl]-4-yl- (2.66%) were the main oxygenated monoterpenes, and the diterpene hydrocarbons were mainly represented by m-Camphorene (5.43%). Six EO samples of R. lionadioides collected in location exhibited similar chemical composition evidencing a chemical homogeneity. The agar disc diffusion method showed that R. lonadioides EO was effective against Bacillus cereus, Pseudomonas aeruginosa, and Staphylococcus aureus with diameters of inhibition ranging from 17±2.65 to 12.33±1.13 mm, and the minimum inhibitory concentration (MIC) values were between 4 and 10 µL /mL. The lowest MIC values (4.0 µL/mL) were measured for Bacillus cereus.