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<i>Acanthamoeba</i> genus is a widely distributed and opportunistic parasite with increasing importance worldwide as an emerging pathogen in the past decades. This protozoan has an active trophozoite stage, a cyst stage, and is dormant and very resistant. It can cause <i>Acanthamoeba</i> keratitis, an ocular sight-threatening disease, and granulomatous amoebic encephalitis, a chronic, very fatal brain pathology. In this study, the amoebicidal activity of sixteen <i>Laurencia</i> oxasqualenoid metabolites and semisynthetic derivatives were tested against <i>Acanthamoeba castellanii</i> Neff. The results obtained point out that iubol (<b>3</b>) and dehydrothyrsiferol (<b>1</b>) possess potent activities, with IC₅₀ values of 5.30 and 12.83 &#181;M, respectively. The hydroxylated congeners thyrsiferol (<b>2</b>) and 22-hydroxydehydrothyrsiferol (<b>4</b>), active in the same value range at IC₅₀ 13.97 and 17.00 &#181;M, are not toxic against murine macrophages; thus, they are solid candidates for the development of new amoebicidal therapies.