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Bovine mastitis, an inflammation of the mammary glands, is mainly caused by bacteria such as Staphylococcus aureus. While antibiotics are the primary treatment for this disease, their effectiveness is often diminished due to resistant strains and biofilm formation, creating the need for safer and more efficient therapies. Plant-based oil therapies, particularly those derived from the genus Eugenia, are gaining popularity due to their pharmacological potential and historical use. In this study, we evaluated the antibacterial, antibiofilm, and synergistic potential of essential oils (EOs) from four species of the genus Eugenia (E. brejoensis, E. gracillima, E. pohliana, and E. stictopetala) against S. aureus isolates from bovine mastitis. The EO of E. stictopetala was obtained by hydrodistillation, and its composition was analyzed using gas chromatography coupled with mass spectrometry. The experiment employed seven clinical isolates from mastitis and two control strains: ATCC 33591 (methicillin-resistant S. aureus - MRSA) and ATCC 25923 (methicillin-susceptible and biofilm producer). A broth microdilution assay was used to determine the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of the EOs and oxacillin. The EO of E. stictopetala contained (E)-caryophyllene (18.01%), β-pinene (8.84%), (E)-nerolidol (8.24%), and α-humulene (6.14%) as major compounds. In the MIC assay, all essential oils showed bactericidal and bacteriostatic effects, especially the species E. brejoensis and E. pohliana, which had MICs ranging from 64 to 256 µg/mL. Regarding the antibiofilm effect, all essential oils were capable of interfering with biofilm formation at subinhibitory concentrations of ½ and ¼ of the MIC. However, they did not significantly affect pre-established biofilms. Additionally, a synergistic interaction was detected between the EOs and oxacillin, with a reduction of 75-93.75% in the antimicrobial MIC. Molecular docking studies indicated that the phytochemicals β-(E)-caryophyllene, (E)-nerolidol, Δ-elemene, and α-cadinol present in the EOs formed more stable complexes with penicillin-binding proteins, indicating a possible mechanism of antibacterial action. Therefore, these results show that the essential oils of Eugenia spp. are promising sources for the development of new therapeutic methods, opening new perspectives for a more effective treatment of bovine mastitis., Competing Interests: Declarations. Consent for publication: All authors agree to publish this work. Ethical approval: This article did not contain any studies with animals performed by any of the authors. Consent to participate: Not applicable Conflict of interest: On behalf of all authors, the corresponding author states that there is no conflict of interest., (© 2024. The Author(s) under exclusive licence to Sociedade Brasileira de Microbiologia.)