Despite a major threat to the public health in tropical and subtropical regions, dengue virus (DENV) infections are untreatable. Therefore, efforts are needed to investigate cost-effective therapeutic agents that could cure DENV infections in future. The NS2B-NS3 protease encoded by the genome of DENV is considered a critical target for the development of anti-dengue drugs. The objective of the current study was to find out a specific inhibitor of the NS2B-NS3 proteases from all four serotypes of DENV. To begin with, nine plant extracts with a medicinal history were evaluated for their role in inhibiting the NS2B-NS3 proteases by Fluorescence Resonance Energy Transfer (FRET) assay. Among the tested extracts, Punica granatum was found to be the most effective one. The metabolic profiling of this extract revealed the presence of several active compounds, including ellagic acid, punicalin and punicalagin, which are well-established antiviral agents. Further evaluation of IC values of these three antiviral molecules revealed punicalagin as the most potent anti-NS2B-NS3 protease drug with IC of 0.91?±?0.10, 0.75?±?0.05, 0.42?±?0.03, 1.80?±?0.16?μM against proteases from serotypes 1, 2, 3 and 4, respectively. The docking studies demonstrated that these compounds interacted at the active site of the enzyme, mainly with His and Ser residues. Molecular dynamics simulations analysis also showed the structural stability of the NS2B-NS3 proteases in the presence of punicalagin. In summary, this study concludes that the punicalagin can act as an effective inhibitor against NS2B-NS3 proteases from all four serotypes of DENV.Communicated by Ramaswamy H. Sarma.