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 IC 50 values of 3 and 11 were 32.58 and 27.82 μM, respectively.