Three new putative sequences of 14‐residue peptaibols, named amazonins I–III were characterized from the endophytic fungus Trichoderma amazonicum via genome mining, high‐performance liquid chromatography coupled to high‐resolution tandem mass spectrometry (LC–MS/MS), and molecular networking. Bioinformatic analysis of the T. amazonicum genome assembly revealed 63 clusters of biosynthetic genes (BGCs) related to secondary metabolites, including a nonribosomal peptide synthetase accountable for the biosynthesis of the discovered peptide sequences. Analysis of the adenylation domains, along with manual interpretation of MS/MS spectra, allowed extensive annotation of the new peptaibol sequences. The combination of bioinformatic tools and LC–MS/MS provides a better opportunity to characterize and identify new peptaibol sequences. Thus, the importance of studies on the production and characterization of peptaibols produced by Trichoderma species from the Amazon region is highlighted.