Staphylococcus aureus is a Gram-positive opportunistic pathogen and a top-priority bacterium in the fight against antimicrobial resistance. Its high propensity to mutate, develop resistance, and become more virulent, as well as its ability to form biofilms, results in difficult-to-treat infections against which new chemical classes are urgently needed. Here, we investigated the antibacterial activity of oxadiazolone-core derivatives (OX) against planktonic and biofilm-associated S. aureus. Among the tested compounds, MpPPOX exhibits a strong bactericidal effect on extracellular bacteria with similar MIC to that of Vancomycin; iBPOX mainly inhibits intracellular bacterial growth; while HPOX strongly impair biofilm formation. Such a divergence in activity prompted us to identify their potential target enzymes via activity-based protein profiling combined with mass spectrometry. Although the most active MpPPOX inhibitor targets multiple (Ser/Cys)-based enzymes, the antibiofilm HPOX compound primarily reacts with enzymes involved in biofilm formation and virulence. Among them, the FabH protein has been confirmed as a vulnerable target of MpPPOX. Overall, this study underscores the multi-target nature of the OXs covalently bind to several (Ser/Cys)-based enzymes of interest. This binding property makes them highly versatile chemotypes that could be used as broad-spectrum antimicrobial and anti-virulence agents to potentiate otherwise ineffective or poorly active drugs.