Background: Due to the complexities of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), an effective medicinal treatment protocol for this lethal disease with a high prevalence has not been approved yet. This study aimed to explore the efficacy of the main alkaloids of Isatis indigotica, one of the richest plant sources of alkaloids against SARS-CoV-2 targets computationally. Materials and Methods: 3D structures of the target proteins including 3CLpro; PLpro, and RdRp were downloaded from Protein Data Bank. The structures of ligands were retrieved from PubChem database or optimized by ORCA program. Ritonavir, Lopinavir, Sofosbuvir, and Remdesivir were selected as control inhibitors. Docking calculations were performed by AutoDock Vina option and top-ranked compounds were subjected to molecular dynamics simulation by Gromacs 5.1.4 simulation package. Result: The results showed that all 15 compounds had stronger interactions with PLpro in comparison to the other enzymes. Dihydroxylisopropylidenylisatisine A binds to the active site of PLpro with highest affinity (-9.3 kcal/mol) which is even more than the binding constants of Ritonavir and Lopinavir. Of the 15 compounds, Dihydroxylisopropylidenylisatisine A and Isatibisindosulfonic acid B had the highest tendency to bind to 3CLpro. Dihydroxylisopropylidenylisatisine A, Indirubin, Insatindibisindolamide A, Indigo, Insatindibisindolamide B, Isatibisindosulfonic acid B and Isatindosulfonic acid B had the highest RdRp binding affinity even more Remdesivir. Conclusion: Based on the results, the highest and weakest interaction with all three enzymes was observed for Dihydroxylisopropylidenylisatisine A and Epigoitrin, respectively. Based on these findings, Dihydroxylisopropylidenylsatistine A might be potential therapeutic candidate against SARS-CoV-2. © 2022 Wolters Kluwer Medknow Publications. All rights reserved.