Smilax ovalifolia Roxb. is a medicinal herb in southern Thailand. However, there are not many studies on active compounds from this plant. This study aimed to optimize extraction conditions for total phenolic content (TPC) from S. ovalifolia root using microwave-assisted extraction (MAE) and to determine the phytochemical compositions and antioxidant activity of the extract. The result showed that the optimal conditions of phenolic compounds extraction include ethanol concentration of 85% v/v, a solid-to-solvent ratio of 1:30 (g/mL), microwave power of 450 W, and extraction time of 150 s. The phenolic-rich extract exhibited strong antioxidant activity with IC50 of 6.31 ± 0.05 mg/mL. LC-MS was used to fingerprint analysis of the extract. The result revealed the presence of 18 bioactive compounds. The main components of S. ovalifolia root extract were some flavonoids, including catechin, epicatechin, procyanidin B2, and quercetin.