The non-structural 5B (NS5B) polymerase of hepatitis C virus (HCV) is an attractive target for antiviral intervention. Quercetagetin (Que) is a natural flavonoid, which has been exhibited to have anti-HCV property through inhibition of RNA binding to NS5B. The last few decades have witnessed a growing interest in the extraction of natural flavonoids with a plethora of different biological activities. Considering the high therapeutic potential of Que, the aim of this study is to explore wide structure entities with potent activity using Que as a prototype. A virtual screen protocol involving docking and molecular dynamics has been performed to examine the potency of forty-three natural flavonoids which recently extracted from plants for inhibition of NS5B. During two screening stages, two compounds 24 and 41 were identified to have more favorable binding affinity to NS5B as compared to Que. The comparative analysis showed that there is a significant difference in the binding free energy of Que and 41 (ΔΔG = -11.17 kcal/mol). It was revealed that van der Waals (vdW) interaction drives the binding process of both 24 and 41 and plays an important role in increasing their activities relative to Que. PHE162 serves as a crucial residue in both the NS5B-24 and NS5B-41 systems, contributing the most vdW energy by π-π interaction, suggesting that aromatic interactions are critical for the binding of 24 and 41 to NS5B. Moreover, hydrogen bond analysis indicates that the hydrogen bonds formed by LYS98, THR137, ASP164 and ARG168, can play important roles in the increased binding affinity of 41 to NS5B relative to Que. The findings of this study will provide useful structure-activity relationship (SAR) guidelines for the design of novel inhibitors with improved/enhanced therapeutic activities in the treatment of hepatitis C.