Abstract Considering the therapeutic potential of medicinal plants as alternatives to antibiotic therapy, the research aims to evaluate the antimicrobial activity of Commiphora leptophloeos against isolates of Staphylococcus spp. from the milk of ruminants with subclinical mastitis. For this, the crude ethanolic extract from the bark and leaves of Commiphora leptophloeos was prepared, with these being chemically characterized by HPLC-DAD-MS and by MALDI-TOF. The extracts were then evaluated as to their antimicrobial effects against 60 isolates of Staphylococcus spp. through the broth microdilution technique to determine the minimum bactericidal concentration. In addition, the extracts were evaluated as to their ability to interfere with biofilm formation and with the already established biofilm. Although all tested extracts showed antimicrobial action, lower MBC values were recorded for the bark extract in the concentration 781.2 µg/mL (25/60). The extracts of the bark and leaves were able to interfere with the initial stages of biofilm formation, but there was no interference of the extract on the established biofilm. There was a high sensitivity of Staphylococcus spp. isolates from subclinical mastitis cases in ruminants when subjected to the extracts from bark and leaves of Commiphora leptophloeos, as well as regarding the ability of extracts to interfere in biofilm formation, indicating their potential in the use for ruminant mastitis therapy.