A novel antibacterial ointment using bacitracin, specific for Gram-positive bacteria, and gramicidin S, a highly toxic antibacterial peptide, was here developed showing broad-spectrum antibacterial activities against pathogenic strains with less toxicity after self-assembly into nanofiber structures. Such structures were confirmed with scanning electron microscopy and CD analyses. In addition, in silico studies using docking associated with molecular dynamics were carried out to obtain information about fiber structural oligomerization. Thus, the bacitracin and gramicidin S-based self-assembled nanopeptide ribbon may be a successful ointment formulation for bacterial infection control.