Trophozoites and cysts of 20 isolates of Acanthamoeba from the cornea and five from related samples were tested in vitro for sensitivity to ten drugs (three aromatic diamidines, two aminoglycosides, two macrolides, a polyene macrolide antibiotic, an organoarsenical and an antimetabolite) and two cationic antiseptics (chlorhexidine and polyhexamethylene biguanide, PHMB). Only chlorhexidine and PHMB showed uniform amoebacidal activity. Aromatic diamidines (pentamidine isethionate, propamidine isethionate and diminazene aceturate) generally proved effective against both forms of the amoeba; only pentamidine gave synergy with the biguanide while propamidine gave an additive effect. Other drugs tested proved erratic or ineffective against different isolates. Chlorhexidine alone, or together with propamidine, was subsequently used in two patients with proven Acanthamoeba keratitis; the causative isolates were sensitive to the individual compounds and to the combination in vitro. The treatment provided resolution of the clinical disease; amoebae were shown to be nonviable by histology and culture. The combination of chlorhexidine and propamidine is recommended for treatment of proven Acanthamoeba keratitis.