The World Health Organization recently reported an alarming evolution and spread of antibiotic resistance, a global risk factor recognized as a One Health challenge. In veterinary, the general lack of clear treatment guidelines often leads to antibiotic misuse. Bovine mastitis is responsible for major economic losses and the main cause of antibiotic administration in the dairy industry, favoring the emergence of multi-resistant phenotypes. The complexity of inter-microbial and host-pathogen interactions in the mammary gland, demonstrated by culture-independent techniques, not only complicates the prediction of antibiotic treatment outcomes but also underscores the urgent need for further research in this field. This work evaluated the interactions between S. aureus L33 and Enterobacter sp. L34 obtained from an intramammary co-infection. The behavior of the dual-species culture resembled that of the Enterobacter monoculture in all the evaluated contexts. Most of the selected S. aureus virulence factors and the antibiotic susceptibility were altered by coexisting with Enterobacter. Under the protection of Enterobacter, S. aureus was able to survive upon exposure to concentrations of cloxacillin and other antibiotics that would be bactericidal for the monoculture. This could have serious implications for bacterial clearance of mastitis originating from the underestimated co-infections. These findings highlight the importance of broadening our knowledge of how microbial interactions in intramammary infections could contribute to antibiotic treatments failures. Moreover, they open new perspectives for the design of bovine mastitis therapies that consider the ecological context in order to optimize the antibiotic usage, improve the success rates and reduce antibiotic resistance.