A set of structurally related diphenylurea derivatives bearing aminoguanidine moiety were synthesized, and their antibacterial activity was assessed against a panel of multi-drug resistant Gram-positive clinical isolates. Two compounds 6 and 24 were identified with better bacteriological profile than the lead compound I. The multi-step resistance development studies indicated that MRSA are less likely to develop resistance toward diphenylurea compounds. Moreover, these compounds demonstrated a prolonged post-antibiotic effect than that of vancomycin. Furthermore, compounds 6 and 24 were able to re-sensitize VRSA to vancomycin, resulting in 8- to more than 32-fold improvement in vancomycin MIC values against clinical VRSA isolates. Finally, when assessed in an in vivo skin infection mouse model, the efficacy of compound 24 was very comparable to that of the commercially available fusidic acid ointment. Additionally, the diphenylurea 24 did not have a pronounced effect on the animal weights along the experiment indicating its safety and tolerability to mice. Taken together, these results indicate that the diphenylurea scaffold merits further investigation as a promising anti-staphylococcal treatment option.