Aculeacin A, a new antifungal antibiotic was isolated from the mycelial cake of Aspergillus aculeatus M-4214. The antibiotic is a white amorphous powder soluble in lower alcohols and hardly soluble in other organic solvents or water. Aculeacin A gave palmitic acid and five ninhydrin-positive products including theonine, hydroxyproline upon acid hydrolysis. The antibiotic showed a potent activity against molds and yeasts, but exhibited no antibacterial activity. Aculeacin A has relatively low toxicity in mice.