Marine biofouling on the surface of ship hull causes the hydrodynamic drag, fuel increase, and higher costs. In this case antifouling paint is often used to protect the submerged surface from marine organisms. In traditional antifouling paints, toxic biocides have been banned due to their severe environmental pollution. To develop novel environmental benign antifouling agents has been our pursuit, and among them, active substances from marine organism for antifouling application are our research focus. In the paper, a natural material in halobios was isolated from a kind of algae Ulva pertusa, which is the most common green alga. The antifouling activities of their extracts were screened by bioassays with two kinds of fouling organisms, diatom and mussel. The crude extract of ethyl acetate was found to be most active against diatom. Silica gel column chromatography (SGCC) and high performance liquid chromatography (HPLC) were used to further isolate the extract of ethyl acetate. In the SGCC extraction, four elution bands were collected, and minimum inhibitory concentrations (MIC) against diatom were determined. Among the four bands, the lowest MIC is 0.7 mg/mL, related to the band YC-EA. The YC-EA band was separated into four parts, and the second part (EE2) showed an inhibitive effect on the settlement of diatom and mussel. The natural product of EE2 can be characterized by a coupled liquid chromatography-mass spectrometry (LC-MS). Results demonstrated that there are antifouling active substances among extracts of Ulva pertusa. Compounds would be a potentially natural antifouling material, resulting in higher standards of environmental safety with outstanding antifouling performance.