Objectives: Securigera securidaca Degen & Dorfl (Fabaceae) is an annual herb occurring wild in West Asia, Europe and Africa. The seeds of this plant are used for the treatment of disorders such as hyperlipidemia, diabetes, and epilepsy in Iranian folk medicine. Materials: This study was carried out to check antiherpetic substances of crude methanol seed extract of Securigera securidaca and its column chromatographic fractions. The antiherpetic activities of different concentrations (20, 2, 0.2 and 0.02 μg/ml) of crude methanol extract and sub-fractions were tested by use of plaque-forming unit (PFU) assay and the real-time polymerase chain reaction (PCR) assay. Results: The most active fractions analyzed by column chromatography, containing kaempferol and kaempferol- 7-O-glucoside. The apparent effective concentration for 50% plaque reduction (EC50) of crude methanol extract, kaempferol, kaempferol-7-O-glucoside and acyclovir (ACV) were 2, 0.2, 0.1 and 0.1 μg/mL, respectively. Conclusion: These findings demonstrate that kaempferol and kaempferol-7-O-glucoside isolated from S. securidaca may inhibit virus attachment to the cell membrane, virus entry into the cell and viral polymerase