Orthosiphon stamineus Benth is a dietary supplement and traditional Chinese herb with widespread clinical applications, but a comprehensive understanding of its active compounds and polypharmacological mechanisms is lacking. This study aimed to systematically investigate the natural compounds and molecular mechanisms of O. stamineus via network pharmacology.Information on compounds from O. stamineus was collected via literature retrieval, while physicochemical properties and drug-likeness were evaluated using SwissADME. Protein targets were screened using SwissTargetPrediction, while the compound-target networks were constructed and analyzed via Cytoscape with CytoHubba for seed compounds and core targets. Enrichment analysis and disease ontology analysis were then carried out, generating target-function and compound-target-disease networks to intuitively explore potential pharmacological mechanisms. Lastly, the relationship between active compounds and targets was confirmed via molecular docking and dynamics simulation.A total of 22 key active compounds and 65 targets were identified and the main polypharmacological mechanisms of O. stamineus were addressed. The molecular docking results suggested that nearly all core compounds and their targets possess good binding affinity. In addition, the separation of receptor and ligands was not observed in all dynamics simulation processes, whereas complexes of orthosiphol Z-AR and Y-AR performed best in simulations of molecular dynamics.This study successfully identified the polypharmacological mechanisms of the main compounds in O. stamineus, and predicted five seed compounds along with 10 core targets. Moreover, orthosiphol Z, orthosiphol Y, and their derivatives can be utilized as lead compounds for further research and development. The findings here provide improved guidance for subsequent experiments, and we identified potential active compounds for drug discovery or health promotion.Copyright © 2023 The Authors. Published by Elsevier Ltd.. All rights reserved.