Infections caused by pathogenic bacteria have been responsible for a significant number of deaths in recent decades. Invasive infections caused by Staphylococcus aureus are highly prevalent and have high morbidity and mortality rates. Faced with the increase in multidrug-resistant bacteria, a promising strategy is the development of adjuvant molecules that enhance the effects of antibiotics, such as efflux pump inhibitors. Betulinic acid (BA) is a pentacyclic triterpene of the lupane type, found in various plant species, which has shown various pharmacological activities, including antibacterial potential. This study investigated the inhibitory action of BA on the MepA efflux pump in strains of Staphylococcus aureus K2068, as well as carrying out fluorescence and membrane permeability tests. The minimum inhibitory concentrations (MIC) were determined using the broth microdilution method. Subsequently, their effects on efflux pump-mediated antibiotic resistance were evaluated by reducing the MIC of the antibiotic and ethidium bromide (EtBr), while fluorimetry and permeability potential tests were carried out using the SYTOX Green fluorescence method. BA did not show intrinsic antibacterial activity, however it showed synergistic effects when associated with the antibiotics ciprofloxacin and ethidium bromide, inducing a reduction in MIC and indicating inhibitory effects on the MepA efflux pump. BA also induced a significant increase in fluorescence and demonstrated the ability to permeabilize the bacterial membrane. The results obtained show that BA has a high potential to act as an efflux pump inhibitor and could help in the treatment of resistant bacterial infections.