6-Hydroxyflavone is a naturally occurring flavone found in the leaves of Barleria prionitis, a plant species in the Acanthaceae family native to India that is widely used against neurological disorders such as paraplegia, sciatica, etc. 6-Hydroxyflavone partially potentiated GABA-induced currents in native GABAA receptors expressed in cortical neurons via BZ site, as the enhancement was blocked by the antagonist flumazenil. Furthermore, in patch clamp studies, 6-Hydroxyflavone displayed the significant preference for a2- and a3- containing subtypes, which were thought to mediate anxiolytic effect, compared to a1- and a5- containing subtypes expressed in HEK 293T cells. In mice, 6-Hydroxyflavone exhibited the anxiolytic-like effect in the elevated plus-maze test, unaccompanied at anxiolytic doses by the sedative, cognitive impairing, myorelaxant, motor incoordination and anticonvulsant effects commonly associated with classical BZs when tested in the hole-board, step-through passive avoidance, horizontal wire, rotarod, and pentylenetetrazol (PTZ)-induced seizure tests, respectively. The findings, therefore, identified 6-Hydroxyflavone as a promising drug candidate for the treatment of anxiety-like disorders.