Amoebiasis, caused by Entamoeba histolytica, remains one of the most important protozoan infections worldwide and continues to be a major public health problem, particularly in developing countries where poor sanitation and unsafe drinking water facilitate disease transmission. Although metronidazole is the drug of choice for the treatment of amoebiasis, its prolonged use is associated with adverse effects, treatment failures, and concerns regarding the emergence of drug resistant strains, emphasizing the need for safer and more effective alternative therapies. Medicinal plants represent an important source of structurally diverse bioactive compounds with significant antiparasitic potential. The present study aimed to comparatively evaluate the in vitro antiamoebic activity and cytotoxicity of leaf and rhizome extracts of Cyperus rotundus L. prepared using ethanol, methanol, ethyl acetate, and chloroform. Antiamoebic activity against Entamoeba histolytica trophozoites was determined using a 96-well microplate assay at concentrations ranging from 20–100 µg/mL, with metronidazole serving as the reference drug. Cytotoxicity was evaluated on L929 mouse fibroblast cells using the MTT assay. All extracts exhibited concentration dependent antiamoebic activity. Among the leaf extracts, the ethyl acetate extract demonstrated the highest activity, producing 58.98% inhibition at 100 µg/mL with an IC₅₀ value of 91.02 µg/mL. Among the rhizome extracts, the methanolic extract exhibited the greatest antiamoebic efficacy, producing 66.11% inhibition with an IC₅₀ value of 70.95 µg/mL. Cytotoxicity studies revealed that all extracts maintained more than 60% viability in L929 fibroblast cells at the highest tested concentration, indicating relatively low toxicity toward normal mammalian cells. Notably, the methanolic rhizome extract demonstrated the most favorable balance between antiamoebic activity and cytocompatibility. The findings provide scientific evidence supporting the traditional medicinal use of Cyperus rotundus and suggest that its rhizome is a promising source of bioactive compounds for the development of novel plant-derived antiamoebic agents. Further investigations involving bioassay-guided fractionation, isolation and characterization of active constituents, mechanistic studies, molecular docking, pharmacokinetic evaluation, and in vivo validation are warranted to establish its therapeutic potential.