Antibiotic resistance represents a widespread problem in milk production. The identification of compounds for a topically applied ointment used in mastitis therapy remains elusive. Compounds from the genus Hymenaea can be administered in cases of multi-drug-resistant Staphylococcus aureus infection for ruminant species, but the protective properties are not well known. Wi this research the aim is verify the protective effects of H. martiana against S. aureus infection in bovine mammary epithelial cell line (MAC-T) and to obtain an antioxidant profile evaluation in vitro. The MAC-T cells were challenged with S. aureus after being exposed to the extract of the H. martiana in the protective assay. For the verification of the viability of the MAC-T cells, the MTT assay was performed, and was used dilutions of the plant extract, starting at 2.5%. The extract of H. martiana was evaluated for antioxidant aspect in different diluition by FRAP, ORAC and DPPH. A variety of flavonoids (quercetin, luteolin, etc.) have been identified as the main components by using mass spectrometry, reinforcing our in vitro findings that flavonoids, especially quercetin, have a medicinal profile capable of killing mastitis-causing bacteria. An excellent antioxidant pattern was observed in the 2.5% solution; however, membrane integrity in MAC-T cells was compromised. Those findings suggest low dilutions of H. martiana extract has a desirable protective effect from S. aureus pathogenesis. Our in vitro studies can be gleaned upon for further in vivo studies.