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<i>Pachyrhizus erosus</i> (L.) Urb. is a tropical perennial vine plant native to southern Mexico, Southeast Asia, Central America, and Africa. In this study, we analyzed and identified various polyphenolic compounds and isoflavones present in ethanolic <i>P. erosus</i> root extracts and investigated their potential anti-obesity activity as a natural health food resource. The extraction process involved drying the yam bean, followed by extraction with 70% ethanol, evaporation, and freeze-drying. Fractionation was achieved through layer separation using <i>n</i>-hexane, ethyl acetate (EtOAc), butanol (BuOH), and water. The EtOAc fraction exhibited the highest antioxidant activity among the experimental groups, with an IC₅₀ value of 531.77 µg/mL for ABTS radical scavenging. In α-glucosidase and lipase inhibition assays, IC₅₀ values were determined to be 873.07 µg/mL and 915.02 µg/mL, respectively. Using HPLC and LC-MS/MS, we detected isoflavone components in <i>P. erosus</i> root extracts, identifying daidzein, genistein, and rotenone among them. Daidzein was the most abundant isoflavone in <i>P. erosus</i> root extracts. To validate the anti-obesity activity in the EtOAc fraction and daidzein, we used 3T3-L1 preadipocytes treated with MDI (3-isobutyl-1-methylxanthine, dexamethasone, insulin) for 8 days. Oil Red O staining experiments demonstrated a concentration-dependent reduction in lipid content in the EtOAc fraction and daidzein treatment groups. Additionally, we examined the expression pattern of proteins related to the leptin-PPAR-FAS Pathway, revealing a concentration-dependent decrease in obesity-related proteins.