To investigate the chemical constituents of Lepidium latifolium L. and explore its antioxidant potential, this study conducted phytochemical isolation and bioactivity evaluation of the n-butanol extract from the plant. The separation and purification of L. latifolium were carried out using silica gel column chromatography, MCI gel column chromatography and HPLC. The structures of the isolated compounds were elucidated based on NMR and HR-MS. To assess antioxidant activity, the ferric reducing antioxidant power (FRAP) assay was employed to determine the total antioxidant capacity of the compounds, and molecular docking was used to evaluate their binding affinities to antioxidant-related targets. A total of five glycosidic compounds were isolated, including a new flavonol tetraglycoside, lepidiumoside (1), identified for the first time from this plant, along with four known glycosides: 2, 3-dihydroxybenzoic acid 3-O-glucoside (2), quercetin 3-O-sophoroside-7-O-rhamnoside (3), quercetin 3-O-β-D-glucopyranoside-7-O-β-D-glucopyranosyl-(1→3)-α-L-rhamnopyranoside (4) and gentisic acid 5-O-β-D-glucoside (5). Among them, compound 3 exhibited the strongest antioxidant activity (the FRAP value is 1.60 ± 0.35 mmol/L), significantly surpassing the positive controls quercetin and vitamin C (with FRAP values of 0.22 ± 0.06 and 0.06 ± 0.05 mmol/L, respectively). Molecular docking further revealed that compound 3 binds favorably to antioxidant-related targets, including Kelch-like ECH-associated protein 1 (KEAP1), superoxide dismutase 1 (SOD1), and ceruloplasmin (CP), demonstrating strong target recognition ability. This study enhances the understanding of the chemical constituents of L. latifolium and provides theoretical support for its potential application in antioxidant-related fields.