Calotropis gigantea is widely used in traditional medicine across rural and tribal regions for treating various ailments. The safety profile of this plant especially in concerning long term or high dose exposure, remains inadequately studied. This study aims to scientifically assess the toxicity of C. gigantea stem extract using zebrafish (Danio rerio) as a model organism. Chronic exposure over 10, 20 and 30 days revealed significant oxidative stress, mitochondrial impairment and histopathological alterations in vital organs. Key antioxidant enzymes glutathione reductase (GR), glutathione S-transferase (GST), succinate dehydrogenase (SDH), catalase (CAT) and superoxide dismutase (SOD) were analysed in the liver, gills, brain and muscles. Enzyme activity has initially increased but declined by the 30th day, indicating progressive oxidative damage. The liver and gills exhibited the most substantial biochemical and structural changes. The histological analysis confirmed cellular degeneration, inflammation and necrosis. These findings highlight the potential risks associated with unregulated therapeutic use of C. gigantea and emphasize the need for scientific validation and public awareness to ensure safe application.