Alternaramide (1), a cyclic pentadepsipeptide, has been isolated from an EtOAc extract of the marine-derived fungus Alternaria sp. SF-5016 by various chromatographic methods. The structure of 1 was mainly determined by analysis of the NMR spectroscopic data, along with chemical methods such as Marfey's and Mosher's methods. Alternaramide (1) showed weak antibiotic activity against Bacillus subtilis and Staphylococcus aureus.