The high morbidity and mortality rates associated with invasive methicillin-resistant Staphylococcus aureus (MRSA) infections underscore the pressing need to develop novel antibiotics. ClpX, functioning as a cochaperone protein of ClpP, has been identified as a crucial target in combating MRSA. In this study, we screened an in-house library and identified a small molecule compound, PFK-158, with high affinity for Staphylococcus aureus ClpX (Kd = 4.1 μM). Simultaneously, PFK-158 displayed potent activity against MRSA (MIC = 2 μg/mL, MBC = 8 μg/mL). Further optimization resulted in a novel α, β-unsaturated ketone bearing a quinolinyl group and a 2-bromophenyl substituent with comparable binding affinity for Staphylococcus aureus ClpX (Kd = 3.6 μM), and it showed enhanced antibacterial activity against MRSA and lower propensity for inducing resistance. Significantly, the novel α, β-unsaturated ketone bearing a quinolinyl group and a 2-bromophenyl substituent demonstrated favorable in vivo safety, oral bioavailability (F = 37.9 %), and promising therapeutic effects in a MRSA-infected skin abscess model. Overall, our findings presented a novel SaClpX modulator with the potential to combat Staphylococcus infections, and suggested a promising strategy for the further development of specific Staphylococcus aureus ClpX modulators.Copyright © 2025 Elsevier Masson SAS. All rights reserved.