Guanylate-binding proteins (GBPs) are a family of interferon-inducible GTPases that are critical components of innate immunity. We recently found that mouse GBP1, in addition to the previously reported GBP2 and GBP5, targets and kills intracellular Francisella novicida, and protects mice from F. novicida infection. These observations suggest that GBPs carry specific antimicrobial features that could be harnessed into therapies to combat bacterial infections. To test this hypothesis, we interrogated the sequence of GBP1 to predict regions that have high antimicrobial probability. We synthesised four putative antimicrobial peptides (GBP128-67, GBP1209-238, GBP1424-452, GBP1558-577; superscripted number indicates the amino acid position) and investigated their ability to kill F. novicida. Remarkably, GBP128-67 exhibited antimicrobial activity against F. novicida based on colony-forming unit (CFU) assays and flow cytometry (IC50 of 1.1 µM). Confocal microscopy revealed that FITC-tagged GBP128-67 localised to the bacterial membrane and resulted in the release of cytoplasmic DNA suggesting bacteriolysis. Electron microscopy confirmed that GBP128-67 treated F. novicida had a marked loss of membrane integrity and expulsion of intracellular content. Furthermore, we show that GBP128-67 is specific for bacteria and does not induce toxicity in mammalian cells. Lastly, we investigated the antimicrobial spectrum GBP128-67. Using CFU assays, we examined the viability of a range Gram-positive and Gram-negative bacteria following GBP128-67 treatment and found that GBP128-67 exhibited antimicrobial activity against Moraxella catarrhalis and Neisseria meningitidis, but not against Citrobacter rodentium, Escherichia coli, Pseudomonas aeruginosa, Shigella flexneri, Salmonella Typhimurium, Bacillus cereus, Listeria monocytogenes, and Staphylococcus aureus. Collectively, our study identified that GBP128-67 is a highly pathogen-selective, and non-toxic antimicrobial that could be developed into a bona fide therapy for bacterial infections. Our findings provide evidence that innate immune proteins such as GBPs are a source of antimicrobial peptides that can be used to develop novel therapies to help combat the growing issue of antimicrobial resistance.