Five Iranian Trichoderma isolates from species T. viride, T. viridescens, T. asperellum, T. longibrachiatum and T. citrinoviride — selected from the Fungal Collection of the Bu Ali Sina University, Hamedan, Iran — were investigated for their peptaibol production. All examined isolates showed remarkable antibacterial activities during the screening of their extracts for peptaibol content with a Micrococcus luteus test culture. HPLC-ESI-IT MS was used for identification and elucidation of the amino acid sequences of peptaibols. The detected peptaibol compounds contain 20 or 18 amino acid residues and belong to the trichobrachin and trichotoxin groups of peptaibols, respectively. T. longibrachiatum and T. citrinoviride produced trichobrachins, while trichotoxins could be detected in T. viride, T. viridescens and T. asperellum. Out of 37 sequences detetermined, 26 proved to be new, yet undescribed compounds, while others were identified as previously reported trichotoxins (trichotoxin A-50s and T5D2) and trichobrachins (longibrachins AI, AII, AIII, BII and BIII). Compounds within the two groups of detected peptaibols differed from each other only by a single or just a few amino acid changes.