The fungal strain Alternaria alternata JS0515 was isolated from Vitex rotundifolia (beach vitex). Twelve secondary metabolites, including one new altenusin derivative (1), were isolated. The isolated metabolites included seven known altenusin derivatives (2–8), two isochromanones (9, 10), one perylenequinone (11), and one benzocycloalkanone (12). Their structures were determined via 1D and 2D nuclear magnetic resonance (NMR) spectroscopy, mass spectrometry (MS), and computational electronic circular dichroism (ECD) analysis. Compounds 3 and 11 increased pyruvate dehydrogenase (PDH) activity in AD-293 human embryonic kidney cells and significantly inhibited PDH phosphorylation. The IC50 values of 3 and 11 were 32.58 and 27.82 μM, respectively.