The effective transportation of antibiotics to bacteria embedded within a biofilm consisting of a dense matrix of extracellular polymeric substances is still a challenge in the treatment of bacterial biofilm associated infections. Here, we developed an antibiotic nanocarrier constructed from quaternary ammonium-tethered hyperbranched polyureas (HPUs-QA), which showed high loading capacity for a model antibiotic, rifampicin, and high efficacy in the transportation of rifampicin to biofilms. The rifampicin-loaded HPUs-QA nanoassembly (HPUs-Rif/QA) demonstrated a synergistic antimicrobial effect in killing planktonic bacteria and eradicating the corresponding biofilms. Compared to the treatment of bacteria-infected chronic wounds by either HPUs-QA or rifampicin alone, HPUs-Rif/QA showed superior efficacy in promoting wound healing by more effectively inhibiting bacteria colonization. This study highlights the potential of the HPUs-QA nanoassembly in synergistic action with antibiotics for the treatment of biofilm associated infections.