The emergence of new infections and increase of bacteria drug-resistance rise up the urgent need for the development of new antibacterial agents from natural sources. This study was designed to evaluate the antibacterial activity of the crude methanolic extract (CME) and fractions [hexane (H), dichloromethane (DCM), ethyl acetate (EA) and ethanol (Et)] obtained from Larrea tridentata (Sessé & Moc. Ex DC.) Coville leaves. The antibacterial activity was determined by the agar diffusion method against six strains of Gram-positive and Gram-negative bacteria. The micro-dilution method was applied for the determination of the minimal inhibitory concentration (MIC) of selected bacteria strains. HPLC analyses of tested samples were also carried out. The antibacterial activity of the samples was more effective inhibiting the growth of Gram-positive bacteria comparing with Gram-negative bacteria, mainly for the CME, DCM and EA fractions. EA fraction showed the highest antibacterial activity against methicillin-resistant Staphylococcus aureus isolated from secretion; with a MIC value (31.3 μg/mL) lower than the reference antibiotic tetracycline (64 μg/mL). Low MIC values (62.5 μg/mL) were also obtained for the CME and DCM fraction. CME and EA fraction presented the highest concentrations of quercetin, kaempferol and nordihydroguaiaretic acid. These compounds have important biological activities and could be responsible for at least part of the antibacterial activity of the CME, DCM and EA fractions. EA fraction from L. tridentata leaves was the most efficient to inhibit the growth of the bacterial strain methicillin-resistant S. aureus, which represents an important step for the search and development of a new antibacterial agent.