Acanthamoeba keratitis remains very difficult to treat because of the lack of antiamoebic agents completely effective against cysts. Currently, the recommended treatment includes the use of topical neomycin sulfate, various imidazoles, and propamidine isethionate (Brolene) 0.1% eye drops, a compound of the diamidine family. In the present article, we describe the successful management of two patients with an Acanthamoeba keratitis, treated with hexamidine diisethionate (Desomedine) 0.1% eye drops, another diamidine derivative, which was found amoebicidal in vitro on the isolated Acanthamoeba strains. This is to our knowledge the first report on the amoebicidal effectiveness of hexamidine, simultaneously in vitro and in vivo.