We investigated the effect of Arogh, a polyherbal formulation (PHF) on animal models of anxiety based on exploratory behavior. The anxiolytic activity of polyherbal formulation (30, 100, 300 and 500 mg/kg) was studied using various behavioural paradigms such as elevated plus maze (EPM), light/dark apparatus (LDA), open field apparatus (OFA), hole board apparatus (HBA). Diazepam (1 mg/kg) was used as a standard anxiolytic drug. The effect of PHF (100 and 300 mg/kg) on serotonin, dopamine and noradrenaline mediated behaviour was studied by lithium induced head twitches in rats, haloperidol induced catalepsy in mice and clonidine induced hypothermia in rats respectively. In EPM, PHF (100, 300 and 500 mg/kg) significantly (P < 0.05) increased the time spent in open arms and the number of entries in open arms. In LDA, PHF (100, 300 and 500 mg/kg) significantly (P < 0.05) increased the time spent in lit zone. In OFA, PHF (100, 300 and 500 mg/kg) significantly (P < 0.05) increased the number of assisted rearing and the number of squares traversed. In HBA, PHF (100, 300 and 500 mg/kg) significantly (P < 0.05) increased the number of head poking. In lithium induced head twitches, PHF (100 and 300 mg/kg) significantly (P < 0.05) decreased the number of head twitches. In haloperidol induced catalepsy, PHF (300 mg/kg) decreased the duration of catalepsy significantly (P < 0.05) at 60 min. In clonidine-induced hypothermia, PHF (300 mg/kg) did not modify the effect. Drugs must be carefully assessed on EPM test and therefore in the present study EPM is supported by other tests. Present study indicates that Arogh, a polyherbal formulation possess anxiolytic activity. It diminished serotonergic transmission and decreased the duration of catalepsy indicating potentiation of dopaminergic transmission. Thus, Arogh a polyherbal formulation contains bioactive principles which possess anxiolytic activity and modified 5-HT and DA mediated behaviour.