Malaria infection caused by Plasmodium falciparum is majorly responsible for millions of deaths in humans every year. Moreover, a rapid increase in resistance to existing drugs has posed an urgent need for new anti-malarials. Herein, we report the highly potent anti-malarial activity of benzopyrano(4,3-b)benzopyran derivatives, inspired from naturally occurring dependensin against chloroquine (CQ) sensitive and resistant P. falciparum strains. Chemically synthesized, four dependensin analogs 85(A–D) exhibited growth inhibition at nanomolar concentrations ranging from 63.96 to 725.8 nM by blocking the parasite development at the ring and early trophozoite stages. The growth inhibitory activity of dependensin analogs was correlated with their anti-plasmodial lactate dehydrogenase activity by computational analysis. Molecular docking, 50 ns simulation and a 2D-Quantitative Structure-Activity Relationship (2D-QSAR) modelling revealed the interaction with their putative target P. falciparum lactate dehydrogenase (PfLDH). Here, developing the predictive 2D descriptors such as thermodynamic, spatial, electronic, and topological with multiple linear regression analysis (MLRA), the structural requirements for potent and selective PfLDH inhibitory activity has been identified. The strong binding of compound 85D to the catalytic Nicotinamide adenine dinucleotide (NADH) binding pocket of the PfLDH further supported the PfLDH targeting potential of dependensin analogs. Overall, this study revealed a highly potent anti-malarial activity of benzopyrano(4,3-b)benzopyran derivatives with their putative anti-PfLDH activity. Communicated by Ramaswamy H. Sarma. © 2020 Informa UK Limited, trading as Taylor & Francis Group.