Introduction: Anacyclus pyrethrum is used in Ayurveda to treat various ailments, ranging from toothache to female infertility. However, the molecular mechanisms of its medicinal properties are obscure and need exploratory studies. Method: We aimed to investigate the molecular mechanisms associated with the medicinal properties of A. pyrethrum using zebrafish as an in vivo model. We have employed in silico, in vitro, and in vivo approaches to elucidate the anti-inflammatory, antioxidant, and cytoprotective functions of the methanolic extracts of A. pyrethrum. In silico analyses were carried out using Prediction of Activity Spectra for Substances (PASS), Molecular Inspiration, Swiss Absorption, Distribution, Metabolism, Excretion and Toxicity-ADME (SWISSADME), Absorption, Distribution, Metabolism, Excretion, and Toxicity-Structure-Activity Relationship(ADMETSar) and molecular docking tools to prove the anti-inflammatory effects of the bioactive components of A. pyrethrum. Results: Our results indicate that the bioactive components of A. pyrethrum have pharmaceutical properties with high intestinal absorption and greater bioavailability. Further, in vitro and in vivo results support the anti-inflammatory, antioxidant, and antitoxic effects of methanolic extract of A. pyrethrum via suppressing the levels of the inflammatory and Reactive oxygen species biomarkers, including Lactate Dehydrogenase, Myeloperoxidase, Nitric Oxide, and Lipid peroxidase respectively. Conclusion: The results obtained from three different approaches in this study highlight the potential anti-inflammatory, antitoxic, and antioxidant effects of A. pyrethrum. © 2025 Elsevier B.V., All rights reserved.