The clinical use of antimicrobials is essential for the treatment of infectious diseases. However, indiscriminate use and/or incorrect prescription makes the scenario of bacterial resistance even more complex. In clinical treatments, the combination of antimicrobials and corticosteroids is used to modulate the inflammatory process triggered by the infection and prevent adverse reactions from antimicrobials, such as nephrotoxicity. Several combinations are available as topical formulations, and they are commonly prescribed for simultaneous intravenous administration in hospitals. Notwithstanding eventual benefits, the safety of these combinations remains poorly investigated. The aim of the present study is to conduct in vitro tests to assess the interference of hydrocortisone on the antimicrobial activity of some drugs against clinical isolates of Staphylococcus aureus and to provide insights of the chemical nature of such interactions. Tests of minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) were performed with and without the addition of hydrocortisone (HYDRO). HYDRO elevated MIC and MBC of oxacillin, and abrogated the activity of gentamicin and chloramphenicol, suggesting that these combinations may pose a risk to clinical treatments. More studies should be carried out in animal models to confirm these effects in vivo.