ETD151, an analogue of the antifungal insect defensin heliomicin, is an antifungal peptide active against yeasts and filamentous fungi. To decipher the mechanisms underlying its molecular action on the phytopathogenic fungus Botrytis cinerea, a necrotrophic pathogen responsible for gray mold disease, we investigated the changes in 3 day-old mycelia upon treatment with different concentrations of ETD151. Optical and fluorescence microscopies were used prior to establishing the peptide/protein profiles through two mass spectrometry approaches: MALDI profiling, to generate molecular mass fingerprints as peptide signatures, and a gel-free bottom-up proteomics approach. Our results show that a concentration of ETD151 above the half-maximal inhibitory concentration can alter the integrity of the mycelial structure of B. cinerea. Furthermore, reproducible modifications of the peptide/ protein composition were demonstrated in the presence of ETD151 within a 1500−16,000 mass (m/z) range. After the robustness of LC-ESI-MS/MS analysis on B. cinerea mycelial extracts was confirmed, our analyses highlighted 340 significantly modulated proteins upon treatment with ETD151 within a 4.8−466 kDa mass range. Finally, data mapping on KEGG pathways revealed the molecular impact of ETD151 on at least six pathways, namely, spliceosome, ribosome, protein processing in endoplasmic reticulum, endocytosis, MAPK signaling pathway, and oxidative phosphorylation.