Salusin-β is an endogenous bioactive peptide that was identified in a human full-length enriched cDNA library using bioinformatics analyses. In our previous study, we found that synthetic salusin-β exhibits antibacterial activity against only Gram-positive microorganisms such as Staphylococcus aureus NBRC 12732. Salusin-β has an ability to depolarize the cytoplasmic membrane of this bacterium, and this phenomenon may be linked to the antibacterial activity of this peptide. A cell-penetrating peptide (CPP), human immunodeficiency virus (HIV)-1 transactivator of transcription (Tat) (49-57) is a short cationic peptide that can traverse cell membranes. In this report, synthetic peptide conjugates of salusin-β and HIV-1 Tat(49-57) showed potent antibacterial activities against both Gram-positive Staphylococcus aureus NBRC 12732 and Gram-negative Escherichia coli NBRC 12734. The synthetic peptides also depolarized the cytoplasmic membrane of Escherichia coli NBRC 12734 as well as Staphylococcus aureus NBRC 12732. These results suggested that HIV-1 Tat(49-57) is a protein transduction domain or CPP that changes the interaction mode between salusin-β and the cell membrane of Escherichia coli NBRC 12734. By binding to HIV-1 Tat(49-57), salusin-β showed a broad antibacterial spectrum regardless of whether the target was a Gram-positive or Gram-negative bacterium.