Background and purpose: The MreB protein plays a crucial role in bacterial cell wall synthesis. The cellular function of MreB can be specifically inhibited by the antibacterial agent A22, which was found to be effective only against Gram-negative bacteria. The antibiotic vancomycin is used for infections caused by Gram-positive bacteria, as it cannot penetrate easily through the outer lipid layer of Gram-negative bacteria. The goal of this study was to describe the effect of A22 on the localisation of vancomycin labelling pattern. Experimental Approach: We treated Escherichia coli cells with Bodipy-vancomycin and we examined how the lifetime and localization of fluorophore fluorescence changed with A22 treatment. We determined the steady-state anisotropy of Bodipy-vancomycin in the presence of MreB. Molecular docking calculations identified the amino acids involved in MreB-vancomycin binding. Fluorescence spectroscopy and microscopy and sedimentation studies have supported the computational results. Microbiological assays were performed on E. coli cells in the presence of A22 and vancomycin. Key Results: The MreB protein directly binds the vancomycin. The A22 facilitates the entry of vancomycin into Gram-negative cells. Vancomycin alters the conformation of MreB and promotes intermolecular interaction between the monomers. A22 and vancomycin interact synergistically to inhibit MreB function. Conclusions and Implications: We identified a new target of the antibiotic vancomycin in the bacterium, which is the MreB protein. The binding of vancomycin leads to structural and functional modifications in the MreB molecule. When used in combination with A22, vancomycin may be able to inhibit cell wall synthesis also in Gram-negative bacteria.