Species of the genus Lippia are rich in essential oils and have shown antibacterial properties, which may be related to oils’ chemical composition. The present work aimed to evaluate the antimicrobial potential of Lippia origanoides Kunth against two bacteria strains: Escherichia coli and Staphylococcus aureus. Leaf essential oils were obtained by hydrodistillation in a modified Clevenger-type apparatus, and their chemical composition was determined by gas chromatography coupled to mass spectrometry (GC/MS) and flame ionization detection (GC/FID). We identified 28 compounds, representing 98.87% of the total concentration of the essential oil. The compounds identified at the highest concentrations were 1,8-cineole (35.04%), carvacrol (11.32%), p-cymene (8.53%), α-pinene (7.17%), and γ-terpinene (7.16%). The leaf essential oil of L. origanoides showed antibacterial action on biological isolates of Escherichia coli and Staphylococcus aureus. For Escherichia coli, the oil presented bactericidal action at concentrations of 5–20 μL/mL. Regarding Staphylococcus aureus, the bactericidal effect was noted at 20 μL/mL and the bacteriostatic action was noted around 2.5–10 μL/mL. Given the results obtained, L. origanoides essential oil showed promising biological potential against Gram-positive (Staphylococcus aureus) and Gram-negative (Escherichia coli) bacteria, thus encouraging further studies on substances isolated from this species to contribute to the development of new antimicrobial drugs.