The aim of the present study was to assess the in vivo potential of therapy based on Hymenaea martiana Hayne extract in mastitis cases among goats that were experimentally induced with strains of Staphylococcus aureus. The phytochemical characterization of the plant extract was performed using HPLC–DAD. Ten female goats (20 mammary glands) were experimentally infected with a clinical strain of S. aureus. The experimental infection was performed in four groups, each consisting of five mammary glands. One group was treated with a commercial antimicrobial and another with ointment from natural plant extract. The other two groups were controls. The diagnosis of mastitis was performed using a bacterial culture, SCC, CFU/mL and CMT. The animals were monitored at nine experimental periods. Three milk samples were subjected to chromatography to survey the constituents present in the ointment prepared with the plant extract. Phytochemical screening of crude ethanol extract confirmed the presence of phenolic compounds, flavonoids, steroids and terpenoids. The results of the HPLC confirmed the presence of phenolic compounds in the chloroform and ethyl acetate. Concerning SCC and CFU/mL, the analysis of variance performed between the experimental periods confirmed that there was a higher score at the time of the first collection after infection (P1) in all of the experimental groups. After treatment with ointment containing the extract of H. martiana and commercial antimicrobials, these variables returned to values indicative of an absence of intra-mammary infection, based on the international standards for raw goat's milk. In the present study, milk collected on the 32nd day post treatment with ointment containing the extract of H. martiana exhibited a significant reduction in CFU/mL, when compared with the control group. Antibacterial activity may be related to the classes of secondary metabolites found.