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Introduction: This study describes the molecular profile and the potential antiviral activity of extracts from Phyllanthus brasiliensis, a plant widely found in the Brazilian Amazon. The research aims to shed light on the potential use of this species as a natural antiviral agent., Methods: The extracts were analysed using liquid chromatography-mass spectrometry (LC-MS) system, a potent analytical technique to discover drug candidates. In the meantime, in vitro antiviral assays were performed against Mayaro, Oropouche, Chikungunya, and Zika viruses. In addition, the antiviral activity of annotated compounds was predicted by in silico methods., Results: Overall, 44 compounds were annotated in this study. The results revealed that P. brasiliensis has a high content of fatty acids, flavones, flavan-3-ols, and lignans. Furthermore, in vitro assays revealed potent antiviral activity against different arboviruses, especially lignan-rich extracts against Zika virus (ZIKV), as follows: methanolic extract from bark (MEB) [effective concentration for 50% of the cells (EC 50 ) = 0.80 μg/mL, selectivity index (SI) = 377.59], methanolic extract from the leaf (MEL) (EC 50  = 0.84 μg/mL, SI = 297.62), and hydroalcoholic extract from the leaf (HEL) (EC 50  = 1.36 μg/mL, SI = 735.29). These results were supported by interesting in silico prediction, where tuberculatin (a lignan) showed a high antiviral activity score., Conclusions: Phyllanthus brasiliensis extracts contain metabolites that could be a new kick-off point for the discovery of candidates for antiviral drug development, with lignans becoming a promising trend for further virology research., (© 2023 John Wiley & Sons Ltd.)