In light of the biomedical significance of metallo-β-lactamases (MβLs), ten new mercaptoacetic acid thioester amino acid derivatives were synthesized and characterized. Biological activity assays indicated that all these synthesized compounds are very potent inhibitors of L1, exhibiting an IC50 value range of 0.018-2.9 μM and a K i value range of 0.11-0.95 μM using cefazolin as substrate. Partial thioesters also showed effective inhibitory activities against NDM-1 and ImiS with an IC50 value range of 12-96 and 3.6-65 μM, respectively. Also, all these thioesters increased susceptibility of E. coli cells expressing L1 to cefazolin, indicated by a 2-4-fold reduction in MIC of the antibiotic. Docking studies revealed potential binding modes of the two most potent L1 inhibitors to the active site in which the carboxylate group interacts with both Zn(II) ions and Ser221. This work introduces a highly promising scaffold for the development of metallo-β-lactamase L1 inhibitors.