Bacterial RNA polymerase (RNAP) represents a validated target for the development of broad-spectrum antibiotics. However, the medical value of RNAP inhibitors in clinical use is limited by the prevalence of resistant strains. To overcome this problem, we focused on the exploration of alternative target sites within the RNAP. Previously, we described the discovery of a novel RNAP inhibitor class containing an ureidothiophene-2-carboxylic acid core structure. Herein, we demonstrate that these compounds are potent against a set of methicillin-resistant Staphylococcus aureus (MRSA) strains (MIC 2-16 μg mL(-1)) and rifampicin-resistant Escherichia coli TolC strains (MIC 12.5-50 μg mL(-1)). Additionally, an abortive transcription assay revealed that these compounds inhibit the bacterial transcription process during the initiation phase. Furthermore, the binding mode of the ureidothiophene-2-carboxylic acids was characterized by mutagenesis studies and ligand-based NMR spectroscopy. Competition saturation transfer difference (STD) NMR experiments with the described RNAP inhibitor myxopyronin A (Myx) suggest that the ureidothiophene-2-carboxylic acids compete with Myx for the same binding site in the RNAP switch region. INPHARMA (interligand NOE for pharmacophore mapping) experiments and molecular docking simulations provided a binding model in which the ureidothiophene-2-carboxylic acids occupy the region of the Myx western chain binding site and slightly occlude that of the eastern chain. These results demonstrate that the ureidothiophene-2-carboxylic acids are a highly attractive new class of RNAP inhibitors that can avoid the problem of resistance.