<jats:title>Abstract</jats:title><jats:p>The production of peptaibols by a marine‐related <jats:italic>Trichoderma longibrachiatum</jats:italic> strain was studied using electrospray ionisation multiple‐stage ion trap mass spectrometry (ESI‐MS<jats:sup>n</jats:sup>‐IT) and gas chromatography/electron impact mass spectrometry (GC/EI‐MS). Two major groups of peptaibols were identified, those with long sequences (20 amino acids) and others with short sequences (11 amino acids). This paper describes the methodology used to establish the sequences of short peptaibols in a mixture without previous individual separation. Nine peptaibols were identified. Among them, eight are new, namely as trichobrachin A I–IV (Aib<jats:sub>9</jats:sub>‐Pro<jats:sub>10</jats:sub> sequence) and as trichobrachin B I–IV (Val<jats:sub>9</jats:sub>‐Pro<jats:sub>10</jats:sub> sequence). Original Pro<jats:sub>6</jats:sub>‐Val<jats:sub>7</jats:sub> and Val<jats:sub>9</jats:sub>‐Pro<jats:sub>10</jats:sub> sequences have to be noted. Copyright © 2006 John Wiley & Sons, Ltd.