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Resistant bacterial infections represent one of the major threats in worldwide health services. In this scenario, plant essential oils are considered promising antimicrobial agents. Therefore, this study aimed to evaluate the antimicrobial potential of Pectis substriata essential oil alone and in combination with antibiotics, against clinical drug-resistant bacterial strains. The essential oil from the plant aerial parts was obtained by hydrodistillation. Antimicrobial activity was assessed against standard and clinical bacterial strains by broth microdilution method, and the synergistic effect was evaluated by checkerboard microtiter assay. The oil alone showed significant activity against clinical Staphylococcus warneri (62.5 µg.mL-1), and was moderately active on Staphylococcus aureus (standard strain) and clinical Staphylococcus intermedius (125 and 250 µg.mL-1, respectively). Synergism was achieved for the combinations of essential oil and ampicillin on S. warneri and of oil and kanamycin on S. intermedius. Additive effects were also observed. This is the first report of the chemical composition of P. substriata essential oil, and the results revealed the presence of compounds with proven antimicrobial activity. The oil proved active against resistant Gram-positive cocci, and showed synergism with antibiotics, revealing its potential use as adjuvant or in the development of new alternative treatments of drug-resistant antimicrobial infections.