Aim: Acanthamoeba cause dangerous diseases in humans such as encephalitis and keratitis as an opportunistic pathogen. Due to the antioxidant, antimicrobial, antifungal, anti-Acanthamoeba and anti-leishmania activities of Artemisia, the aim of this study is investigate the effect of aqueous extract of Artemisia aucheri seed on Acanthamoeba trophozoites and cysts in vitro. Materials and Methods: Acanthamoeba trophozoites and cysts were propagated in appropriate culture medium. Aqueous extract of Artemisia aucheri were prepared at concentrations of 2000, 1000, 500, 250, 125 and 62.5 μg/ml and were added to both protozoa forms (trophozoites and cysts). Then, three techniques including trypan blue, MTT and flowcytometry were used to investigate the effect of this extract on Acanthamoeba trophozoites and cysts. Results: It was found that increasing the time and concentration of aqueous extract of Artemisia aucheri seed significantly reduced the number of live Acanthamoeba trophozoites and cysts (P ≤0.05). At the concentration of 2000 µg/ml the number of live trophozoites was 0% and at the concentration of 62.5 µg/ml the number of live trophozoites was 57.7%. Conclusion: The results of this study showed that the aqueous extract of Artemisia aucheri has anti-acanthamoeba activity and seems to have beneficial pharmacological effects on some diseases and complications caused by Acanthamoeba. Further research is needed to determine this issue.