Epithelial Ovarian cancer is the leading cause of cancer mortality among women all over the world. As chemotherapeutics has many side effects, researchers have focused on the potential use of medicinal plants as natural antitumor agents. Xanthium strumarium studied in this work as an herbal anticancer agent. This study aimed to evaluate the antitumor effect and metabolic alterations caused by the root extract of X. strumarium on human ovarian cancer cell line (A2780cp), using NMR-based metabolomics approaches. Cells were cultured and treated with different concentrations of the ethanolic plant extract. Antitumor activity determined by MTT assay and cell metabolites extracted for NMR spectroscopy. 1H NMR spectroscopy was applied, and outliers were analyzed using multivariate statistical analysis techniques. The extract exhibited antitumor activity against ovarian cancer cells with an IC50 of 6 μg/ml after 48 hours of treatment. The most affected metabolic pathways in the experimental groups were limited to tyrosine metabolism, nucleotide metabolism, fatty acid biosynthesis, and glycerolipid metabolism. Our data showed that the ethanolic root extract of X. strumarium has antitumor activity on the ovarian cancer cells and can affect vital metabolic pathways. However, further studies required to validate this activity.