Antimicrobial peptides (AMPs) are gaining interest as potential therapeutic agents. Peptides derived from bovine lactoferricin B (LfcinB) have been reported to exhibit antimicrobial activity, and the LfcinB RRWQWR sequence is the smallest known motif that exhibits antibacterial and cytotoxic activity. Our goal was to examine the effect of multicopy arrangements of the RRWQWR motif, on its antibacterial activity against healthcare-associated infections (HCAIs). Linear and branched peptides containing the RRWQWR motif were generated using solid phase peptide synthesis-Fmoc/tBu methodology, purified, and characterized using reverse phase-high performance liquid chromatography and matrix-assisted laser desorption/ionization time of flight mass spectrometry. For each peptide, the antibacterial activity against Staphylococcus aureus (ATCC 25923 and 33591 strains) and Klebsiella pneumoniae (ATCC 13883 and 700603 strains) was assessed by measuring the minimum inhibitory and the minimum bactericidal concentrations, in the exponential phase. Cells were observed by scanning electron microscopy, and the hemolytic activity of the peptides was assessed. The overall results demonstrate that, compared to linear analogues, polyvalent presentation of the RRWQWR motif enhances its antibacterial activity against both Gram-negative and Gram-positive bacteria even on resistant strain.