<jats:title>Abstract</jats:title><jats:p>The bacterium <jats:italic>Paenibacillus larvae</jats:italic> has been extensively studied as it is an appalling honey bee pathogen. In the present work, we screened crude extracts derived from fermentations of <jats:italic>P. larvae</jats:italic> genotypes ERIC I and II for antimicrobial activity, following the detection of four putative secondary metabolite gene clusters that show high sequence homology to known biosynthetic gene clusters for the biosynthesis of antibiotics. Low molecular weight metabolites produced by <jats:italic>P. larvae</jats:italic> have recently been shown to have toxic effects on honey bee larvae. Moreover, a novel tripeptide, sevadicin, was recently characterized from laboratory cultures of <jats:italic>P. larvae</jats:italic>. In this study, paenilarvins, which are iturinic lipopeptides exhibiting strong antifungal activities, were obtained by bioassay‐guided fractionation from cultures of <jats:italic>P. larvae</jats:italic>, genotype ERIC II. Their molecular structures were determined by extensive 2D NMR spectroscopy, high resolution mass spectrometry, and other methods. Paenilarvins are the first antifungal secondary metabolites to be identified from <jats:italic>P. larvae</jats:italic>. In preliminary experiments, these lipopeptides also affected honey bee larvae and might thus play a role in <jats:italic>P. larvae</jats:italic> survival and pathogenesis. However, further studies are needed to investigate their function.