<jats:title>Summary</jats:title><jats:p><jats:styled-content style="fixed-case">A</jats:styled-content>merican foulbrood (<jats:styled-content style="fixed-case">AFB</jats:styled-content>) caused by the bee pathogenic bacterium <jats:styled-content style="fixed-case"><jats:italic>P</jats:italic></jats:styled-content><jats:italic>aenibacillus larvae</jats:italic> is the most devastating bacterial disease of honey bees worldwide. From <jats:styled-content style="fixed-case">AFB</jats:styled-content>‐dead larvae, pure cultures of <jats:styled-content style="fixed-case"><jats:italic>P</jats:italic></jats:styled-content><jats:italic>. larvae</jats:italic> can normally be cultivated indicating that <jats:styled-content style="fixed-case"><jats:italic>P</jats:italic></jats:styled-content><jats:italic>. larvae</jats:italic> is able to defend its niche against all other bacteria present. Recently, comparative genome analysis within the species <jats:styled-content style="fixed-case"><jats:italic>P</jats:italic></jats:styled-content><jats:italic>. larvae</jats:italic> suggested the presence of gene clusters coding for multi‐enzyme complexes, such as non‐ribosomal peptide synthetases (<jats:styled-content style="fixed-case">NRPS</jats:styled-content>s). The products of these enzyme complexes are known to have a wide range of biological activities including antibacterial activities. We here present our results on antibacterial activity exhibited by vegetative <jats:styled-content style="fixed-case"><jats:italic>P</jats:italic></jats:styled-content><jats:italic>. larvae</jats:italic> and the identification and analysis of a novel antibacterially active <jats:styled-content style="fixed-case"><jats:italic>P</jats:italic></jats:styled-content><jats:italic>. larvae</jats:italic> tripeptide (called sevadicin; <jats:styled-content style="fixed-case">S</jats:styled-content>ev) produced by a <jats:styled-content style="fixed-case">NRPS</jats:styled-content> encoded by a gene cluster found in the genome of <jats:styled-content style="fixed-case"><jats:italic>P</jats:italic></jats:styled-content><jats:italic>. larvae</jats:italic>. Identification of <jats:styled-content style="fixed-case">S</jats:styled-content>ev was ultimately achieved by comparing the secretome of wild‐type <jats:styled-content style="fixed-case"><jats:italic>P</jats:italic></jats:styled-content><jats:italic>. larvae</jats:italic> with knockout mutants of <jats:styled-content style="fixed-case"><jats:italic>P</jats:italic></jats:styled-content><jats:italic>. larvae</jats:italic> lacking production of <jats:styled-content style="fixed-case">S</jats:styled-content>ev. Subsequent mass spectrometric studies, enantiomer analytics and chemical synthesis revealed the sequence and configuration of the tripeptide, <jats:styled-content style="fixed-case">D</jats:styled-content>‐Phe‐<jats:styled-content style="fixed-case">D</jats:styled-content>‐<jats:styled-content style="fixed-case">ALa</jats:styled-content>‐Trp, which was shown to have antibacterial activity. The relevance of our findings is discussed in respect to host–pathogen interactions.