The excess use of antibiotics has resulted in the appearance of multidrug resistant pathogens, described as one of the most eminent threats to human health in the 21st century. To address this problem many unique fundamental processes for bacteria are being probed for the development of new antimicrobial drugs. In bacteria signaling via the two-component system is a widespread mechanism, with bacteria typically featuring in excess of 10 such systems per genome. The two-component system consists of a sensor histidine kinase and a response regulator. A conserved two-component system (YycFG aka WalRK) essential for viability has been discovered in most gram-positive pathogens suggesting that inhibition of these systems would lead to cell death. However, targeting a single system can be expected to quickly result in drug resistances. It would thus seem desirable to discover compounds with the ability to inhibit multiple two-component systems simultaneously. Synthetic compounds have been described as potent competitive inhibitors of DNA gyrase and one such compound has entered phase 1 clinical trials. Gyrases share a common catalytic ATP-binding domain with histidine kinases; redesign of these compounds to bind the catalytic domain of the latter thus seemed possible. We utilized available high-resolution structures of the inhibitor/gyrase complex and of a histidine kinase catalytic domain to rationally design novel compounds and probe histidine kinase binding in silico. The most promising in silico hits were synthesized and tested for their ability to inhibit several histidine kinases of Bacillus subtilis. A series compounds were found to be potent inhibitors of all tested histidine kinases but did not inhibit the structuraly distinct ser/thr kinases in vitro. Several of these compounds showed antimicrobial activities against methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant Enterococcus faecalis (VRE) and other Gram-positive pathogens. Hemolytic assays on sheep erythrocytes ruled out that these compounds act by simply lysing cells, a problem encountered with many previously discovered histidine kinase inhibitors. In summary, the discovered compounds are promising leads for the development of new antibacterial drugs.