Studies of the secondary metabolites produced in culture by the plant pathogenic phylloplane fungus Alternaria brassicicola (Schweinitz) Wiltshire (NRRL 1299 = ATCC 6650) have led to the isolation of brassicicolin A (6), a novel isocyanide antibiotic with potent in vitro activity against the gram-positive bacteria Bacillus subtilis and Staphylococcus aureus. Brassicicolin A is a fully acylated derivative of D-mannitol containing two D-α-hydroxyisovaleryl units, two acetyl groups, and two epimerized α-isocyanoisovaleryl units. The structure of brassicicolin A was assigned as 6 primarily through chemical degradation studies and by NMR and mass spectral analysis of its deisocyanation product. This account represents the first report of the α-isocyanoisovaleryl group as a naturally occurring structural unit.