Abstract Background Traditional preparations of the root of Biebersteinia multifida DC (Geraniaceae), a native medicinal plant of Irano-Turanian floristic region, have been used for the treatment of phobias as anxiolytic herbal preparation. Methods We utilized the phobic behavior of mice in an elevated plus-maze as a model to evaluate the anxiolytic effect of the plant extract and bio-guided fractionation was applied to isolate the active compounds. Total root extract, alkaline and ether fraction were administered to mice at different doses 30 and 90 min prior to the maze test. Saline and diazepam were administered as negative and positive controls, respectively. The time spent in open and closed arms, an index of anxiety behavior and entry time, was measured as an index of animal activity. Results The total root extract exhibited anxiolytic effect which was comparable to diazepam but with longer duration. This sustained effect of the crude extract was sustained for 90 min and was even more after injection of 45 mg/kg while the effect of diazepam had been reduced by 90 min. The anxiolytic effect factor was only present in the alkaline fraction and displayed its effect at lower doses than diazepam while pure vasicinone as the previously known alkaloid did not shown anxiolytic effect. The effect of the alkaline fraction was in a dose dependent manner starting at 0.2 mg/kg with a maximum at 1.0 mg/kg. Bio-guided fractionation using a variety of chromatographic methods led to isolation and purification of three coumarin derivatives from the bioactive fraction, including umbelliferone, scopoletin, and ferulic acid. Conclusion For the first time, bio-guided fractionation of the root extract of B. multifida indicates significant sustained anxiolytic effects which led to isolation of three coumarin derivatives with well-known potent MAO inhibitory and anti-anxiety effects. These data contribute to evidence-based traditional use of B. multifida root for anxiety disorders.