Antimicrobial peptides (AMPs) are essential components of the insect innate immune system. Their diversity provides protection against a broad spectrum of microbes and they have several distinct modes of action. Insect-derived AMPs are currently being developed for both medical and agricultural applications, and their expression in transgenic crops confers resistance against numerous plant pathogens. The antifungal peptide metchnikowin (Mtk), which was originally discovered in the fruit fly Drosophila melanogaster, is of particular interest because it has potent activity against economically important phytopathogenic fungi of the phylum Ascomycota, such as Fusarium graminearum, but it does not harm beneficial fungi such as the mycorrhizal basidiomycete Piriformospora indica. To investigate the specificity of Mtk, we used the peptide to screen a F. graminearum yeast two-hybrid library. This revealed that Mtk interacts with the fungal enzyme β(1,3)-glucanosyltransferase Gel1 (FgBGT), which is one of the enzymes responsible for fungal cell wall synthesis. The interaction was independently confirmed in a second interaction screen using mammalian cells. FgBGT is required for the viability of filamentous fungi by maintaining cell wall integrity. Our study therefore paves the way for further applications of Mtk in formulation of bio fungicides or as a supplement in food preservation.