Four new antibiotic peptaibols, named neoatroviridins A (1), B (2), C (3), and D (4), have been isolated from the culture broth of Trichoderma atroviride. Their amino acid sequences were determined mainly by mass spectrometry in combination with NMR studies. The absolute stereochemistry of neoatroviridins was established as L by GC analysis of their acid hydrolysates with derivatization, except for isovaline which was in D. Neoatroviridins, composed of 18 amino acid residues, showed significant membrane-perturbing activity responsible for their antibiotic action, which was comparable to that of alamethicin, a well-known 20-residue peptaibol.