The C-terminal sequences of porcine thrombin encode a series of peptides with the characteristics of net positive charge and hydrophobicity, suggesting antimicrobial potential. In this study, we synthesized truncated C-terminal peptides to explore their antimicrobial potency and structure-activity relationship. The results showed that some peptides exerted antimicrobial activity against Gram-positive and Gram-negative bacteria, with selectivity for microbial membranes. The antimicrobial potency of the peptides increased with the extension of chain length. Considering toxicity to red blood cells, the 21-mer peptide T-6 displayed the highest therapeutic index of 43.4, suggesting its higher cell selectivity. Typical α-helical conformations were observed upon binding to a bacteria-mimicking environment. The derivatives tended to interact preferentially with negatively charged vesicles compared to zwitterionic vesicles. Flow cytometry and electron microscopy revealed that the peptides targeted bacterial cell membranes and disrupted cytoplasmic membrane integrity, thereby causing the release of cellular contents leading to cell death. Peptide-membrane interaction experiments provided evidence that the peptides killed bacteria via a membrane-mediating mechanism. In summary, the C-terminal sequence of porcine thrombin has antimicrobial functions.