Biofouling in aquatic environments have a wide range of detrimental effects on man-made structures and cause economic loss. Current antifouling compounds including Diuron, dichlorofluanid, and Irgarol are toxic and can accumulate in marine environments. Thus, effective and environmentally friendly antifoulants are needed. Six structurally similar compounds were isolated from the brown alga, Sargassum horneri, based on bioactivity-guided isolation by reversed-phased liquid flash chromatography and highperformance liquid chromatography. Six chemical constituents possessing antifouling activities were identified as chromanols consisting of polyprenyl chain by nuclear magnetic resonance and mass spectroscopy. Antifouling activities of these six compounds were determined against representative fouling organisms including a hard fouling organism the mussel Mytilus edulis, a soft fouling macroalga Ulva pertusa, the biofouling diatom Navicula annexa, and the biofouling bacteria Pseudomonas aeruginosa KNP-3 and Alteromonas sp. KNS-8. The compounds could inhibit larvae settlement of mussel M. edulis with an EC 50 of 0.11-3.34 μg mL −1 , spore settlement of U. pertusa zoospores (EC 50 of 0.01-0.43 μg mL −1 ), and the diatom N. annexa (EC 50 of 0.008-0.19 μg mL −1 ). The two biofouling bacteria were sensitive to the tested compounds (minimum inhibitory concentration of 1.68-36.8 and 1.02-30.4 μg mL −1 , respectively). From toxicity tests on juvenile Sebastes schlegelii fish, brine shrimp Artemia salina, and microalga Tetraselmis suecica, S3 had the lowest LC 50 values of 60.2, 108, and 6.7 μg mL −1 and exhibited no observed effect concentration at 24.5, 41.6, and 3.1 μg mL −1 for these three tested marine organisms, respectively.