Inhalation of water contaminated with Naegleria fowleri may lead to a potentially fatal infection of the central nervous system known as primary amebic meningoencephalitis (PAM). Amphotericin B (AMB), an antifungal drug, is the only agent with established clinical efficacy in the treatment of PAM, though therapy with this drug is not always effective and has been associated with adverse effects on the kidneys and other organs. We investigated the activity of various therapeutic agents against N. fowleri in an attempt to identify other useful agents for treating PAM. Several of these agents exhibited in vitro activity against the Lee (M67) strain of N. fowleri. The minimum inhibitory concentrations of these agents were 0.1 μg/ml (ketoconazole), 1 μg/ml (liposomal AMB), and 10 μg/ml (minocycline, quinupristin–dalfopristin, and trifluoperazine). Other agents had a minimum inhibitory concentration >10 μg/ml (linezolid) or >100 μg/ml (rifampin). In a mouse model of PAM, none of the untreated control mice survived, whereas the survival of treated animals was 50% (quinupristin–dalfopristin), 30% (ketoconazole and liposomal AMB), 20% (trifluoperazine), and 10% (linezolid and minocycline). Further studies are needed to ascertain whether these agents have synergistic activity with AMB in vitro and in vivo.