A series of diketo esters and their pertinent bioisosteres were designed and synthesized as potent antibacterial agents by targeting methionine amino peptidases (MetAPs). In the biochemical assay against purified MetAPs from Streptococcus pneumoniae (SpMetAP1a), Mycobacterium tuberculosis (MtMetAP1c), Enterococcus faecalis (EfMetAP1a) and human (HsMetAP1b), compounds 3a, 4a and 5a showed more than 85% inhibition of all the tested MetAPs at 100 μM concentration. Compounds 4a and 5a also exhibited antibacterial potential with MIC values 62.5 μg/mL (S. pneumoniae), 31.25 μg/mL (E. faecalis), 62.5 μg/mL (Escherichia coli) and 62.5 μg/mL (S. pneumoniae), 62.5 μg/mL (E. coli), respectively. Moreover, 5a also significantly inhibited the growth of multidrug resistant E. coli strains at 512 μg/mL conc., while showing no cytotoxic effect towards healthy CHO cells and thus being selected. Growth kinetics study showed significant inhibition of bacterial growth when treated with different conc. of 5a. TEM analysis also displayed vital damage to bacterial cells by 5a at MIC conc. Moreover, significant inhibition of biofilm formation was observed in bacterial cells treated with MIC conc. of 5a as visualized by SEM micrographs. Interestingly, 5a did not cause an alteration in the hemocyte density in Galleria mellonella larvae which is considered in vivo model for antimicrobial studies and was non-toxic up to a conc. of 2.5 mg/mL.