A new class of inhibitors of the two-component regulatory systems (TCS) of bacteria was discovered based on the salicylanilide screening hits, closantel (1) and tetrachlorosalicylanilide (9). A systematic SAR study versus a model TCS, KinA/Spo0F, demonstrated the importance of electron-attracting substituents in the salicyloyl ring and hydrophobic groups in the anilide moiety for optimal activity. In addition, derivatives 8 and 16, containing the 2, 3-dihydroxybenzanilide structural motif, were potent inhibitors of the autophosphorylation of the KinA kinase, with IC50s of 2.8 and 6. 3 µM, respectively. Compound 8 also inhibited the TCS mediating vancomycin resistance (VanS/VanR) in a genetically engineered Enterococcus faecalis cell line at concentrations subinhibitory for growth. Closantel (1), tetrachlorosalicylanilide (9), and several related derivatives (2, 7, 10, 11, 20) had antibacterial activity against the drug-resistant organisms, methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant Enterococcus faecium (VREF).