The protozoon Naegleria fowleri (N. fowleri) is a free-living amoeba that produces primary amoebic meningoencephalitis (PAM), which is an acute and frequently fatal infection of the central nervous system. We characterized the strains of N. fowleri isolated from the cerebrospinal fluid (CSF) of two cases presented in northwestern Mexico. The strains were isolated and cultured in 2% bactocasitone medium. Enflagellation assays, ultrastructural analysis, protein and protease electrophoresis patterns, and PCR were performed as confirmatory tests. Virulence tests were done using in Balb/c mice. Light microscopy analysis of brain tissue showed amoebae with abundant inflammatory reaction and extensive necrotic and hemorrhagic areas. The enflagellation assay was positive and the electron microscopy showed trophozoites with morphologic features typical of the genus. Protein and protease profiles of the isolated strains were identical to the reference strain. Finally, a 1500-bp PCR product was found in all three strains. Based on all the analyses performed, we concluded that the etiologic agent of both PAM cases was N. fowleri. The need for better epidemiological information and educational programs about basic clinical and pathological aspects of free-living amoebae provided by the health authorities are emphasized.