The present study has been designed to evaluate the cytotoxic and anthelmintic activities of crude methanolic extract of whole plant of Parthenium hysterophorus L. The in-vitro cytotoxic activity of methanolic extract was performed by MTT assay method against CHOK 1 (Chinese hamster ovary cell line) and A-549 (Human lung adenocarcinoma epithelial cell line). The crude methanolic extract (25, 50, 150 mg/ml concentration) of Parthenium hysterophorus whole plant was taken for anthelmintic activity against Pheretima posthuma. Effect of inhibition of cell growth showed significant cytotoxicity against A-549 with an IC50 of 183.00±5.8 μg/ml and against CHOK1 with an IC50 of 230±0.00μg/ml. The results obtained from the study indicate good anthelmintic activity against Pheretima posthuma. The present study concluded that the methanolic extract of Parthenium hysterophorus possess potent cytotoxic and anthelmintic activities. Further investigations are in progress to identify the mechanism of cytotoxic effect and anthelmintic activity.