A series of aromatic sulfonyls substituted icariin derivatives was synthesized and their antibacterial activities against S. aureus (including drug-sensitive bacteria and drug-resistant bacteria) were evaluated. Among them, compound 9 exhibited high potency against methicillin-sensitive (MSSA) and resistant strains of S. aureus (MRSA) with MIC values of 1-2 mmol/L. Reverse virtual screening and molecule docking analysis indicated that compound 9 might bind the allosteric site of PBP2a that may inhibit cell wall synthesis, with the advantage of activity against multidrug resistant S. aureus. SPR experiment further confirmed the binding affinity. Therefore, we consider aromatic sulfonyls at C-3 position substituted icariin derivatives to be a novel class of anti-MRSA agents worth of further investigation.