Background: Dengue virus serotype 2 (DENV2) and West Nile virus (WNV) fevers are mosquito-borne diseases with no effective treatment at present. In recent years, the development of plant-based antivirals targeting the viral NS2B-NS3 serine proteases has been the main focus as the synthetic antivirals available are not specific and less safe. Objectives: To evaluate the inhibitory activity of Lepisanthes fruticosa pulp extract against NS2B-NS3 proteases from DENV2 and WNV and identify the metabolites from this fruit extract. Materials and Methods: In vitro DENV2 and WNV NS2B-NS3 proteases assays were carried out using the methanolic extract of L. fruticosa pulp. Liquid chromatography-electron spray ionization-mass spectrometry/mass spectrometry (LC-ESI-MS/MS) and gas chromatography-mass spectrometry/mass spectrometry (GC-MS/MS) were performed to determine the metabolites present in this fruit species extract. Results: L. fruticosa extract exhibited inhibitory activity toward DENV2 and WNV NS2B-NS3 proteases with 50% inhibitory concentration value of 1.733 ± 0.195 and 9.245 ± 0.938 mg/mL, respectively. LC-ESI-MS/MS of L. fruticosa extract identified epigallocatechin-catechin, epigallocatechin, epicatechin, catechin, cyanidin rutinoside, procyanidin trimer, rutin, myricetin rhamnohexoside, luteolin glucoside and its derivative which were from the flavonoid group. In addition, GC-MS/MS identified fatty acids and sterols. Conclusion: The inhibitory activity of L. fruticosa pulp extract toward NS2B-NS3 proteases from DENV2 and WNV suggests this fruit species as a potential source for the development of antiviral. Metabolites from the groups of flavonols, flavones, and sterols identified in L. fruticosa pulp may contribute to the inhibitory properties of L. fruticosa.