INTRODUCTION: A great variety of bioactive natural products has been reported for different Palicourea and Psychotria species (Rubiaceae). However, few of them as well as few of species of these botanical genera have been evaluated for antiplasmodial activity. OBJECTIVE: To assess the antiplasmodial activity of 24 extracts from Palicourea and Psychotria genera, along with the targeted LC-MS metabolite profiling, as well as identification of the main metabolites in the bioactive extracts. METHODS: Twenty four ethanol and acid-base extracts from Palicourea and Psychotria genera collected in the Amazonia and Atlantic Forest, Brazil, were evaluated against chloroquine-resistant Plasmodium falciparum W2 strain by PfLDH. The metabolite profiling and putative identification of metabolites from bioactive extracts were determined by LC-DAD-ESI-MS and LC-HRMS, respectively. RESULTS: The ethanol extracts disclosed low antiplasmodial activity (% GI < 50%). High antiplasmodial effect was observed for the acid-base extracts from Psychotria apoda and Psychotria colorata with 100% inhibition of parasite growth inhibition. Fragment ions related to pyrrolidinoindoline alkaloids were observed by LC-DAD-ESI-MS mainly in the most bioactive extracts. The results of the in vitro screening associated with the LC-DAD-ESI-MS and LC-HRMS(n) data allowed to predict, for the first time, the pyrrolidinoindoline alkaloids as possible antiplasmodial representing, then, new potential natural antimalarial hits. In addition, other metabolite classes such as flavanones, lignans and chalcones were also putatively identified in the bioactive extracts of Psychotria apoda, Psychotria capitata, and Psychotria poeppigiana. CONCLUSION: The present results point to Palicourea and Psychotria species as sources of new antimalarial hits. CI - (c) 2021. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.