Background: A type of N-(hydroxyl) urea-based dipeptides was synthesized as a new class of antibacterial agents based on a naturally occurring hydroxamate-containing dipeptide 3-((1-((2-[Hydroxymethyl]-1-pyrrolidinyl) carbonyl)-2-methylpropyl) carbamoyl) octanohydroxamic acid (actinonin, 1a). Actinonin has been assumed to inhibit bacterial peptide deformylase (PDF), which is a metalloprotease and required for final protein maturation for bacterial survival in all of the bacterial species. Methods: In preliminary screening, N-(hydroxyl) ureido-norleucyl-valyl-indolin-1-ylamide (11c) was two-fold more potent than actinonin against S. aureus. Both N-(hydroxyl) ureido-norleucyl-valyl-(4-benzyl) piperazin-1-ylamide (11a) and 11c also showed moderate potency against B. subtilis and E. coli with MIC at 25 μg/ml. Results: In the bacteria-sensitive test, the potency of 11a and 11c were relatively equal to kanamycin with MIC of 25 μg/ml and 50 μg/ml, respectively. Conclusion: No cytotoxic effects on HEK 293 cells were observed at 10 μM levels for the tested compounds.