Multi-drug resistant bacteria are a great clinical global concern due to the fact that they can't be fought efficiently with conventional antibiotics. Antimicrobial peptides (AMPs) found in the innate immune system of a wide range of organisms might prove useful to fight infections, due to the reported slower development of resistance to AMPs. Increasing the cationicity and keeping moderate hydrophobicity of the AMPs have been described to improve antimicrobial activity. We previously found a peptide derived from the Tribolium castaneum insect defensin 3, exhibiting antrimicrobial activity against several human pathogens. Here, we analyzed the effect against Staphyloccocus aureus of an extended peptide (TcPaSK) containing two additional amino acids, lysine and asparagine, flanking the former peptide fragment in the original insect defensin 3 protein. Through flow cytometry and electron microscopy techniques we observed that the TcPaSK peptide showed greater antimicrobial activity against S. aureus. According to the high antimicrobial activity of TcPaSK peptide against S. aureus cells, SEM and TEM analysis showed structural damage of the plasma membrane, leakage of cytoplasmic debris, and complete breakdown of S. aureus cells challenged with TcPaSK peptide. The potent activity of the TcPaSK peptide as an antimicrobial warrants to explore its use in new therapeutic developments as an alternative approach to overcome bacterial resistance to antibiotics.