Abstract: Background and Objective: The world is still in urgent need of natural compounds to solve many problems related to human health, especially from plants. Therefore the current study aims to assess the pharmacological activity of Thevetia peruviana extract. Materials and Methods: Anticancer against prostate cancer cell lines, antioxidant by Ferric Reducing Antioxidant Power (FRAP) assay and antimicrobial by disc diffusion method activities of T. peruviana (family Apocynaceae) Seed Kernel (SK) and Seed Pericarp (SP) extract were evaluated. Analysis of SK and SP phytoconstituents by GC-MS was performed. Results: Cytotoxic activity of SK and SP extracts were investigated and showed strong cytotoxicity with critical effects on the proliferation at IC50 values 0.05 and 35 μg mL–1, respectively with remarkable morphological changes. The SK and SP extracts displayed antioxidant activity with 55.55 and 18.19 μm mg–1, respectively. SK extract showed the highest antibacterial and anti-yeast activity than SP extract besides the appearance of changes in the ultrastructures of Klebsiella pneumoniae, Bacillus subtilis and Candida albicans exposed to SK extract. Conclusion: The obtained finding suggested that the methanolic extracts of SK and SP are rich in phytoconstituents and considered a promising new source particularly SK with biological activities that contribute to human diseases treatment.