This study evaluated antimicrobial and hemolytic activities of mastoparan-B (MP-B) isolated from the venom of the hornet, Vespa basalis, and its analogs after substituting certain amino acid (aa) residues. MP-B exhibited significantly different antimicrobial activities against bacteria species/strains tested, especially two Escherichia coli strains, Staphylococcus xylosus and Citrobacter koseri at low dosages, and was non-specific against certain Gram-positive and -negative bacteria. Our results indicated that hydrophobicity modification by single aa substitution may enhance their antimicrobial activities. An aa substituted MP-B, viz., MP-B-1, in which Trp substituted for Leu 3 , became more effective than the original peptide at inhibiting or killing the bacterial species tested, especially Klebsiella pneumonia, Salmonella typhimurium, and Salmonella Cholerasuis, and even up to 8 times more effective in some cases. However, MP-B-2 was virtually similar to MP-B against each bacterial species assayed, while MP-B-3 reduced its effectiveness greatly compared to others. On the other hand, MP-B and its analogs were not effective against the beneficial probiotics and they were not hemolytic to erythrocytes at the dosages tested. Our results suggested that MP-B becomes more potent against specific pathogenic bacteria and safe to the probiotics after undergoing appropriate amino acid substitutions.