Screening of traditional medicines has proven invaluable to drug development and discovery. Utilizing activity-guided purification, we previously reported the isolation of a list of flavonoids from the medicinal herb Scutellaria baicalensis Georgi, one of which manifested an affinity for the benzodiazepine receptor (BDZR) comparable to that of the synthetic anxiolytic diazepam (K i =6.4 nM). In the present study, this high-affinity, naturally occurring flavonoid derivative, 5,7,2′-trihydroxy-6,8-dimethoxyflavone (K36), was chosen for further functional and behavioral characterization. K36 inhibited [ 3 H ]flunitrazepam binding to native BDZR with a K i value of 6.05 nM. In electrophysiological experiments K36 potentiated currents mediated by rat recombinant α 1 β 2 γ 2 GABA A receptors expressed in Xenopus oocytes. This potentiation was characterized by a threshold (1 nM) and half-maximal stimulation (24 nM) similar to diazepam. This enhancement was demonstrated to act via the BDZR, since co-application of 1 μM of the BDZR antagonist Ro15-1788 reversed the potentiation. Oral administration of K36 produced significant BDZR-mediated anxiolysis in the mice elevated plus-maze, which was abolished upon co-administration of Ro15-1788. Sedation, myorelaxation and motor incoordination were not observed in the chosen dosage regimen. Structure–activity relationships utilizing synthetic flavonoids with different 2′ substituents on the flavone backbone supported that 2′-hydroxyl-substitution is a critical moiety on flavonoids with regard to BDZR affinities. These results further underlined the potential of flavonoids as therapeutics for the treatment of BDZR-associated syndromes.