A spectrum of enniatins was isolated from Fusarium sp. strain F31 by bioassay-guided isolation directed against Botrytis cinerea. Two new enniatins, J(2) (7) and J(3) (8), were co-isolated and both contained, in addition to three hydroxyisovaleric acid units, N-methylated l-alanine, l-valine, and l-isoleucine units, differing only in their primary sequence. Two other enniatins, named enniatin J(1) (1) and enniatin K(1) (6), each containing two N-Me-l-Val units and one N-Me-l-Ala or alpha-N-Me-l-butyric acid unit, respectively, were isolated for the first time without directed biosynthesis. The enniatin structures were elucidated by spectroscopic and chemical methods, and the absolute configuration of the amino acids (l) and hydroxyisovaleric acid (d) was consistent with all previously isolated enniatins. The known enniatins B (2), B(1) (4), B(2) (5), and B(4) (3) were also isolated. The minimum inhibitory concentration of pure enniatins against Botrytis cinerea was 75 microg/mL.