A broad-spectrum fungicide, calophycin, has been isolated from Calothrix fusca EU-10-1, a terrestrial blue-green alga belonging to Nostocaceae, and identified to be a cyclic decapeptide, cyclic (L-Ala-D-Asp-L-Asn-L-Gln-Gly-L-Arg-N-MeAsn-L-Pro-(2R,3R,4S)-Hamp-L-Val), where Hamp is a (2R,3R,4S)-3-amino-2-hydroxy-4-methylpalmitic acid unit and MeAsn is an N-methylasparagine residue. Its total structure, including absolute stereochemistry, was determined by a combination of spectral and chemical studies, including synthesis of the unusual β-amino acid Hamp. In screening over 1000 strains of laboratory-cultured blue-green algae for fungicidal activity, we found that extracts of more than 10% of these prokaryotes show activity against one or more of five test organisms: Aspergillus oryzae, Candida albicans, Penicillium notatum, Saccharomyces cerevisiae, and Trichophyton mentagrophytes. We report here the isolation and total structure determination of this strongly antifungal cyclic decapeptide from Calothrix fusca strain EU-10-1. In a disc-diffusion assay, calophycin at 1.2 μg/disc showed zones of inhibition of 13, 7, 12, 12, and 15 mm against the five test organisms, respectively. MIC values against C. albicans, T. mentagrophytes, and A. fumigatus were 1.25, 2.5, and 1.25 μg/mL, respectively, compared to amphotericin B's MIC values of 0.625 and 1.25 μg/mL against C. albicans and A. fumigatus. Calophycin was moderately cytotoxic with an IC50 of 0.24 μg/mL against the KB human nasopharyngeal carcinoma cell line.