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Background: Aspergillus flavus is one of the most abundant mold present around the world. The present study was conducted to investigate the acute toxicity, analgesic and sedative effect of the crude extract obtained from soil borne fungi A. flavus. Methods: The fungi was isolated from soil samples and identified morphologically and microscopically. The growth condition i.e. media, temperature, pH, and incubation period were optimized. In these optimized growth condition, A. flavus was grown in batch culture in shaking incubator. Crude contents were extracted by using ethyl acetate solvent. Crude secondary metabolites were screened for acute toxicity, analgesic and sedative effect. Results: Upon completion of the experiment, blood was collected from the tail vein of albino mice, and different haematological tests were conducted. White blood cells counts displayed a slight increase (10.6× 109/L) above their normal range (0.8–6.8 × 109/L), which may be due to the increment in the number of lymphocytes or granulocytes. However, the percentage of lymphocytes was much lower (17.7%), while the percentage of the granulocytes was higher (61.4%) than its normal range (8.6–38.9%). A reduction in the mean number of writhing in the different test groups was caused by the application of the crude ethyl acetate extract through the i.p. route at different doses (50, 100, and 150 mg/kg body weight). The results of our investigation showed the EtOAc extract of A. flavus can cause a significant sedative effect in open field. Conclusion: It was concluded from the present study that the A. flavus has the potential to produce bioactive metabolites which have analgesic and sedative effect.