Pathogenic strains of Acanthamoeba cause keratitis (AK), granulomatous amoebic encephalitis (GAE), amoebic pneumonitis (AP), and skin infection in human and animals. The treatment of an Acanthamoeba infection is invariably very difficult and not always effective, and compounds that are amebicidic or amebistatic are frequently toxic and/or irritating for humans. Squaramides and polyamine derivatives have been demonstrated to have antitumor and antiprotozoal activity. The aim of this study was to investigate the activity of 5 squaramides and 5 acyclic polyamines against trophozoites and cysts of A. castellanii Neff. Amoebicidal activity against the trophozoites and cytotoxicity against Vero cells were evaluated with a colorimetric assay, using Alamar Blue®, and chlorhexidine digluconate was assayed as the reference drug. The squaramides 3 and 5 and the acyclic polyamine 6 appeared to be the most active against the trophozoites and their cytotoxicity was low, showing selectivity indexes of 28.3, 26, and 25.7, respectively, similar to the control drug, chlorhexidine digluconate (27.6). But only the squaramide 3 showed complete cysticidal activity at the concentrations of 100 and 200 µM, as the chlorhexidine digluconate. Further studies of the mechanism of action and in vivo assays are needed, but squaramide 3 could be used for developing novel therapeutic approaches against Acanthamoeba infections.