Phomenoic acid and phomenolactone, previously isolated from the fungus Phoma lingam (Tode) Desm., have shown moderate antifungal and antibacterial properties in vitro. To rationalize the production of phomenoic acid, a kinetic study of its biosynthesis in the mycelium was performed. Phomenoic acid and phomenolactone appear in the mycelium after a prolonged incubation, a phenomenon which may be of particular interest for the production of these substances or in the study of the mechanism of their biosynthesis. The isolation procedure was optimized for phomenoic acid. Through a series of SiO2 column chromatographies, high-pressure liquid chromatography, and transformation of the mixture of phomenoic acid and phomenolactone into methyl phomenoate (BF3), the final yield of phomenoic acid reached 160 mg/liter of culture medium. An alternative method for the isolation of both phomenoic acid and phomenolactone is also reported in detail.