The rise of multidrug-resistant underscores the need for novel therapeutic targets beyond conventional peptidoglycan biosynthesis. Some bacterial strains bypass MurA inhibition by fosfomycin via a cell wall salvage pathway. This study targeted AmgK (AmgK) and MurU (MurU) to identify inhibitors that could complement fosfomycin therapy. A malachite-green-based dual-enzyme assay enabled efficient activity measurements and high-throughput chemical screening. Screening 232 compounds identified Congo red and CTAB as potent MurU inhibitors. A targeted mass spectrometric analysis confirmed the selective inhibition of MurU relative to that of AmgK. Molecular docking simulations indicate that Congo red preferentially interacts with MurU through electrostatic contacts, primarily involving the residues Arg28 and Arg202. The binding of Congo red to MurU was corroborated further using SUPR-differential scanning fluorimetry (SUPR-DSF), which revealed ligand-induced thermal destabilization. Ongoing X-ray crystallographic studies, in conjunction with site-directed mutagenesis and enzyme kinetic analyses, aim to elucidate the binding mode at an atomic resolution.