The aim of this study was to investigate the activity of diosgenin against Naegleria fowleri trophozoites at the cellular and molecular levels. Diosgenin (100 μg/ml; 241.2 μM) had a 100% inhibitory effect on N. fowleri trophozoites (5 x 10(5) cell/ml). Scanning electron micrograph revealed diosgenin decreased the number of sucker-like apparatuses and food cup formation among N. fowleri trophozoites at 3 and 6 hours post-exposure, respectively. Diosgenin down-regulated the nf cysteine protease gene expression of N. fowleri trophozoites at 6 and 12 hours post-exposure. The toxicity to mammalian cells caused by diosgenin at therapeutic dose was less than amphotericin B, the current drug used to treat N. fowleri infections. Our findings suggest diosgenin has activity against the surface membrane and the nf cysteine pro tease of N. fowleri trophozoites. However, the other mechanisms of action of diosgenin against N. fowleri trophozoites require further exploration.