The increase in bacterial resistance to conventional chemotherapy has resulted in a resurgent interest in novel antibacterial agents. Focusing on the transglycosylase enzyme activity of penicillin-binding proteins (a target for overcoming resistant phenotypes) and using moenomycin A (a natural product antibiotic that inhibits transglycosylase, with its disaccharide core retaining cell wall inhibitory activity) as a lead, we developed a general solid-phase synthetic strategy to construct a library of moenomycin disaccharide analogues (bypassing the limitations of solution syntheses). A library of 1300 disaccharides was prepared, and six compounds (18-23) were identified that inhibit bacterial cell wall biosynthesis (IC50 < 15 µg/mL) and bacterial growth (MIC < 25 µg/mL). These structurally simpler disaccharides are effective against both antibiotic-sensitive (e.g., Enterococcus faecalis ATCC 29212, Staphylococcus aureus ATCC 29213) and resistant strains (e.g., methicillin-resistant S. aureus, vancomycin-resistant E. faecium/faecalis). They are equipotent to vancomycin in inhibiting cell wall biosynthesis against sensitive Gram-positive bacteria and more effective than vancomycin against resistant Enterococcus. The results support that the entire moenomycin pentasaccharide is not necessary for target recognition and efficacy, and these novel disaccharides represent promising antibacterial agents.