Two new polyacetylene carboxylic acids, petroformynic acids B (3) and C (4), were isolated from a marine sponge Pertrosia sp. as cytotoxic constituents. Their structures were determined by interpretation of 2D NMR data and tandem FABMS data. Absolute stereochemistry of 3 was assigned by application of the modified Mosher analysis. Petroformynic acids exhibit moderate cytotoxic activity against P388 cells.