Flavonoids exert antioxidant activity by scavenging free radicals, chelating metals, and modulating antioxidant enzymes. The root extract of Glycine tomentella Hayata (GTE), a traditional Chinese medicinal herb contains flavonoids, particularly, isoflavones. However, its neuroprotective effects against anxiety remain unclear. In this study, the effects of GTE on anxiety-like behavior and oxidative stress in male Balb/c mice were investigated. The mice were administered GTE orally once daily for 14 d and subsequently, the anxiolytic-like effects of the extract were observed via elevated plus maze and open field tests. Oxidative stress levels in the treated mice were also measured. The results obtained identified daidzein (9.19 mg/g dry extract) and daidzin (2.95 mg/g dry extract) as the key isoflavones in GTE. Furthermore, free radical scavenging assays confirmed that GTE shows strong antioxidant activity, with an IC50 value of 8.82 μg/mL. It also showed pronounced anxiolytic effects, attenuating reactive oxygen species production in a dose-dependent manner. Mechanistic studies attributed these effects to the estrogenic activity of daidzein, which possibly modulates emotional state via estrogen receptor activation and systemic oxidative stress mitigation. These findings suggest that isoflavone-enriched GTE is a natural resource with potential for use as an antioxidant for mitigating anxiety.