AbstractNowadays, an alarming increase in antibiotics resistance have steered intensive research to explore new substitutes for ineffective antibiotics. This study aimed (1) to identify the purified antibacterial peptide isolated from amoebocyte lysate of the Asian horseshoe crab, Tachypleus gigas, (2) to compare potency of antibacterial activity of the purified peptide in comparison to a commercial antibiotic (ampicillin), and (3) to evaluate thermostability and cytotoxicity of the purified peptide. Antibacterial peptide having molecular weight of 7.5 kDa and consisting of 10 amino acid sequence of SYFPGSTYGR at position of 54–63 was identified as tachystatin-A2. Peptide strongly inhibited the growth of 2 bacterial strains known as Escherichia coli ATCC 11775 and Bacillus subtilis ATCC 11774 with MIC values at 0.12 ± 0.09 and 0.12 ± 0.05 µg/mL, respectively. However, for Stapylococcus aureus, the potency of this antibacterial peptide was low, in which the activation only occurred at high concentration (IC50 value = 15.63 ± 0.001 µg/mL). It was thermally stable when heated at 30, 50, 70 and 100°C. The testing cytotoxicity on the human liver cancer cell HepG2 line showed IC50 value of 42.45 µg/mL indicating a non-toxic peptide. Based on its unique properties such as broad-spectrum activity, thermally stable, and non-toxic, the tachystatin-A2 has a huge potential as candidate for antibacterial product.