Aim: To evaluate the effectiveness of Hypocrellins B (HB) photodynamic therapy (PDT) on Acanthamoeba trophozoites and cysts, and its cytotoxic potential on the corneal epithelial cells and stromal cells in vitro. Methods: Acanthamoeba trophozoites and cysts (5x10(4)/ml) were incubated with various concentrations of HB, followed by exposure to light (> 470 nm) for 30 min at a fluence rate of 50 mW/cm(2). Survival rate was assessed using the live/dead viability/cytotoxicity assay and non-nutrient agar-Escherichia coli culture assay. Rabbit corneal epithelial cells and stromal cells were incubated with various concentrations of HB, followed by exposure to the same light fluence rate as above. The corneal cell survival rate was assessed using the live/dead viability/cytotoxicity assay. Results: HB-PDT showed a dose-dependent inhibition on the trophozoites and cysts. The 100% inhibitory concentration (IC100) of HB was 1 mg/ml for the trophozoites and 20 mg/ml for the cysts. The 50% inhibitory concentration (IC50) of HB for the cysts was 3.8 mg/ml and for the trophozoites 0.23 mg/ml. The inhibition of the HB-PDT on the rabbit corneal cells and stromal cells was observed, but HB without irradiation had no inhibition on the corneal cells. Conclusion: HB-PDT had amoebacidal properties in vitro and showed a better effectiveness on the trophozoites than the cysts. Meanwhile, HB-PDT demonstrated cytotoxicity on the corneal epithelial cells and stromal cells, but HB exhibited no appreciable cytotoxicity in the dark to the corneal cells.