A template based on positional residue frequencies in the N-terminal stretch of natural α-helical antimicrobial peptides was used to prepare sequence patterns and to scan the Swiss-Prot Database, using the ScanProsite tool. This search identified a segment in pilosulin 1, a cytotoxic peptide from the venom of the jumper ant Myrmecia pilosula, as a potential novel antimicrobial peptide sequence. This segment, corresponding to the 20 N-terminal residues, was synthesized and its structural properties and biological activities were investigated. It showed a potent and broad spectrum antimicrobial activity including standard and multi-drug resistant gram-positive and gram-negative bacteria and Candida albicans, confirming the validity of the search method. A rational redesign approach resulting in four amino acid substitutions yielded a variant with improved antibacterial and significantly reduced hemolytic activity.