Veronicastrum latifolium (V. latifolium) is rich in biologically active flavonoids, and is widely used in folk medicine. The purpose of this study was to investigate flavonoids of V. latifolium, and evaluate its antioxidant, antimicrobial, and anti-inflammatory activities. Eight flavonoids including vitexin, luteolin, wogonoside, apigenin, hispidulin, chrysoeriol, acacetin, and pectolinarigenin ... [Show full abstract] were identified by LC–MS/MS. The purified extract was obtained by enriching crude extract of V. latifolium through macroporous adsorption resins (MARs). The antioxidant activity of the crude extract and purified extract was evaluated by (1,1-diphenyl-2-picrylhydrazyl) DPPH, (Ferric ion reducing antioxidant power) FRAP, and (2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)) ABTS assay. This study found that the crude extract and purified extract exhibited ability to scavenge free radicals, and the ability of the purified extract to scavenge free radicals was stronger than the crude extract in DPPH, ABTS, and FRAP assay. In addition, the antimicrobial activity of the crude extract and purified extract against five bacterial strains (Staphylococcus aureus (S. aureus), Bacillus subtilis (B. subtilis), Escherichia coli (E. coli), Pseudomonas aeruginosa (P. aeruginosa), and Acinetobacter baumannii (A. baumannii)), and yeast (Candida albicans (C. albicans)) was investigated. The crude extract and purified extract displayed bacteriostatic effects against both gram-positive bacteria and yeast, and the bacteriostatic effects of the purified extract was stronger compared with the crude extract. The vitro cytotoxicity of the crude extract and purified extract against isolated rat peritoneal macrophages (RAW264.7) cells was considered that it was not toxic to RAW264.7 cells at the concentration of 25–300 μg/mL. Anti-inflammatory activity of the crude extract and purified extract was evaluated by their effects on nitric oxide (NO) production in lipopolysaccharide (LPS) induced RAW264.7 cells. The crude extract and purified extract significantly inhibited NO production in RAW264.7 cells at the concentration of 200 μg/mL and 300 μg/mL. This study indicated that V. latifolium has good antioxidant, antimicrobial, and anti-inflammatory effect.