Naegleria fowleri is a ubiquitous, pathogenic free-living amoeba; it is the most virulent Naegleria species and causes primary amoebic meningoencephalitis (PAME) in laboratory animals and humans. Although amphotericin B is currently the only agent available for the treatment of PAME, it is a very toxic antibiotic and may cause many adverse effects on other organs. In order to find other potentially therapeutic agents for N. fowleri infection, the present study was undertaken to evaluate the in vitro and in vivo efficacies of miltefosine and chlorpromazine against pathogenic N. fowleri. The result showed that the growth of the amoeba was effectively inhibited by treatment with amphotericin B, miltefosine, and chlorpromazine. When N. fowleri trophozoites were treated with amphotericin B, miltefosine, and chlorpromazine, the MICs of the drug were 0.78, 25, and 12.5 microg/ml, respectively, on day 2. In experimental meningoencephalitis of mice that is caused by N. fowleri, the survival rates of mice treated with amphotericin B, miltefosine, and chlorpromazine were 40, 55, and 75%, respectively, during 1 month. The average mean time to death for the amphotericin B, miltefosine, and chlorpromazine treatments was 17.9 days. In this study, the effect of drugs was found to be optimal when 20 mg/kg was administered three times on days 3, 7, and 11. Finally, chlorpromazine had the best therapeutic activity against N. fowleri in vitro and in vivo. Therefore, it may be a more useful therapeutic agent for the treatment of PAME than amphotericin B.