Background: BIOGF1K, a compound K-rich fraction prepared from the root of Panax ginseng, is widely used for cosmetic purposes in Korea. We investigated the functional mechanisms of the antiinflammatory and antioxidative activities of BIOGF1K by discovering target enzymes through various molecular studies. Methods: We explored the inhibitory mechanisms of BIOGF1K using lipopolysaccharide-mediated inflammatory responses, reporter gene assays involving overexpression of toll-like receptor adaptor molecules, and immunoblotting analysis. We used the 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay to measure the antioxidative activity. We cotransfected adaptor molecules, including the myeloid differentiation primary response gene 88 (MyD88) and Toll/interleukin-receptor domain containing adaptor molecule-inducing interferon-β (TRIF), to measure the activation of nuclear factor (NF)-kB and interferon regulatory factor 3 (IRF3). Results: BIOGF1K suppressed lipopolysaccharide-triggered NO release in macrophages as well as DPPHinduced electron-donating activity. It also blocked lipopolysaccharide-induced mRNA levels of interferon-β and inducible nitric oxide synthase. Moreover, BIOGF1K diminished the translocation and activation of IRF3 andNF-kB (p50 and p65). This extract inhibited the upregulation ofNF-kB-linked luciferase activity provoked by phorbal-12-myristate-13 acetate aswell asMyD88, TRIF, and inhibitor of kB (IkBα) kinase(IKKβ), and IRF3-mediated luciferase activity induced by TRIF and TANK-binding kinase 1 (TBK1). Finally, BIOGF1K downregulated the NF-kB pathway by blocking IKKβ and the IRF3 pathway by inhibiting TBK1, according to reporter gene assays, immunoblotting analysis, and an AKT/IKKβ/TBK1 overexpression strategy. Conclusion: Overall, our data suggest that the suppression of IKKβ and TBK1, which mediate transcriptional regulation of NF-kB and IRF3, respectively, may contribute to the broad-spectrum inhibitory activity of BIOGF1K.