Fermentation of the marine-derived fungus Trichoderma asperellum, collected from the sediment of the Antarctic Penguin Island, resulted in the isolation of six new peptaibols named asperelines A-F (1-6), which are characterized by an acetylated N-terminus and a C-terminus containing an uncommon prolinol residue. Structures were determined by extensive 1D and 2D NMR ((1)H-(1)H COSY, HMQC, HMBC, NOESY) spectroscopic data analysis combined with ESIMS/MS fragmentation. The absolute configurations of the amino acid residues possessing a chiral alpha-carbon and of the prolinol residue were determined to be L and S, respectively, using a new method of (1)H NMR spectroscopic comparison of complexes formed between the chiral reagent Ru(D(4)-Por*)CO and amino acids derived from the peptaibols with those formed with reference standards.