The antifungal compound AK-3 is purified and characterized from intracellular metabolites of the unicellular cyanobacteria, Synechocystis sp. PCC 6803. AK-3 clearly had antifungal effects upon growth of the fungi Aspergillus spp. From 2 g of dry cell powder, 4.8 mg of AK-3 was obtained with a yield of 0.24%. Its structure was elucidated by NMR, UV spectra, amino acid analysis by chemical degradation, and MS measurements, including MALDI-TOF and MS-MS techniques. AK-3 contains a high level of antifungal activity compared to itraconazole and is seemingly a new compound based on its amino acid composition. Extraction was performed with an acetic acid/water mixture (pH 3.0) while detection at UV 214 nm found AK-3 did not react with ninhydrin. These results suggest that this compound is a cyclic peptide with high hydrophilicity and a phenolic ring.