Novel muraminomicin derivatives with antimicrobial activity against methicillin-resistant Staphylococcus aureus (MRSA) were synthesized by esterification of the hydroxy group on the diazepanone ring of muraminomicin Z1. Compound 1b (DS14450354) possessed a diheptoxybenzyl-β-Alanyl-β-Alanyl group and exhibited minimum inhibitory concentrations (MICs) against MRSA comparable to those against methicillin-susceptible S. aureus (MSSA). The MICs that inhibited 50 and 90% of the strains were 1 and 2 μg/mL, respectively. Compound 1a (DS60182922) possessed an aminoethylbenzoyldodecylglycyl moiety and showed bactericidal activity against MSSA Smith. The bactericidal activity of 1a against MRSA 10925 was comparatively lower, whilst 1b exhibited dose-dependent bactericidal activity against MRSA 10925. The mutation frequency of 1b was lower than that of 1a. An amino acid substitution (F226I) was observed in MraY mutants isolated from culture plates containing 1a or 1b. Subcutaneous 1a and 1b administration showed good therapeutic efficacy in murine systemic infection models with MSSA Smith and MRSA 10925, comparable to that of vancomycin, suggesting that the novel muraminomicin derivatives may be effective therapeutic agents against MRSA that warrant further investigation. A scheme for the formulation of the key ester intermediate, requiring no HPLC preparation, was also established.