Cochliomycin G (1), a new 14-membered resorcylic acid lactone, together with six known analogues (2−7), was isolated from the culture broth of a marine-derived fungus Cochliobolus lunatus. The planar structure of 1 was established by extensive NMR spectroscopy, and the absolute configuration was elucidated by the combination of empirical rules, CD data, and 13C chemical shift calculations. Compound 1 exhibited potent antifouling activity against Chlorella vulgaris, Chaetoceros socialis, and Navicula exigua, with EC50 values of 1.09, 0.92, and 0.61 μg/mL, respectively.