β-Ketoacyl-acyl carrier protein synthase (KAS) III is a condensing enzyme that initiates fatty acid biosynthesis in most bacteria. We determined three pharmacophore maps from receptor-oriented pharmacophore-based in silico screening of the X-ray structure of Escherichia coli KAS III (ecKAS III) and choose 16 compounds as candidate ecKAS III inhibitors. Binding inhibitors were characterized using saturation-transfer difference NMR spectroscopy (STD-NMR), and binding constants were determined with fluorescence quenching experiments. Based on the results, we propose that the antimicrobial compound, 4-cyclohexyliminomethyl-benzene-1,3-diol (YKAs3003), is a potent inhibitor of pathogenic KAS III, displaying minimal inhibitory concentration (MIC) values in the range 128–256μg/mL against various bacteria.