Soybean (Glycine max) is recognized for its nutritional significance due to its high protein and lipid content. The unique presence of isoflavonoids in leguminous plants, particularly soybeans, distinguishes them as promising bioactive compounds. Isoflavonoids, such as genistein, daidzein, and glycitein, are known to exhibit antibacterial properties. However, their potential antiviral activity against plant viruses, specifically ToLCKV, remains largely unexplored. This study aimed to investigate the impact of isoflavonoids on ToLCKV infection in Nicotiana benthamiana, a model plant species. Seeds of N. benthamiana were primed with mixture of genistein, daidzein, glycitein, and water (control). Subsequently, seedlings were inoculated with infectious clones of Tomato leaf curl Karnataka virus (ToLCKV) using the agroinoculation method at 4 leaf stage. Leaves from both primed and unprimed plants were collected for DNA isolation. Real-time PCR was then employed to quantify the abundance of ToLCKV in primed and control plants. The absolute quantification of viral load was assessed and calculated using a standard graph of viral DNA dilutions. The results of the real-time quantification of viral load indicated a 28-fold reduction in the viral load of primed plants compared to the unprimed plants. This observation suggests that isoflavonoids from soybeans, namely genistein, daidzein, and glycitein, possess antiviral activity against ToLCKV. This study provides valuable insights into the potential application of soybean isoflavonoids as natural antiviral agents in agriculture, particularly for the management of ToLCKV infections that pose a substantial threat to pulse crops. Further research is warranted to elucidate the underlying mechanisms of isoflavonoid action and to explore their broader applicability in the field of plant virology.