Three cyclic peptolides have been isolated from two different fungal species and their structures determined. Icosalides A1 (1a), A2 (1b), and B (1c) each contain two serine and two leucine amino acid residues and incorporate two fatty acid moieties as part of the central twenty-member ring. 1a contains L-serine and both D- and L-leucine residues, while 1b and 1c contain only L-amino acid residues. Icosalide A1 displays antimicrobial activity against Streptococcus pyogenes, S. pneumoniae (Felton), and Enterococcus faecalis. Icosalides A2 and B are cytotoxic to replicating MDCK cells.