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A chemical and biological validation of the traditional use of Hyoscyamus niger seeds as anti-inflammatory drug has been established. The methanolic extract of seeds of H. niger (MHN) was evaluated for its analgesic, anti-inflammatory and antipyretic activities in experimental animal models at different doses. MHN produced significant increase in hot plate reaction time, while decreasing writhing response in a dose-dependent manner indicating its analgesic activity. It was also effective in both acute and chronic inflammation evaluated through carrageenin-induced paw oedema and cotton pellet granuloma methods. In addition to its analgesic and anti-inflammatory activity, it also exhibited antipyretic activity in yeast-induced pyrexia model. Furthermore, the bioactive MHN under chemical investigation showed the presence of coumarinolignans as major chemical constituent and yielded a new coumarinolignan …