Dissertation (MSc Chemistry)Antimicrobial resistance could cause approximately 10 million of people's deaths worldwide yearly by 2050 (Sutherland, 2017). Thus, this study was aimed to evaluate the antimicrobial activity of Cleome gynandra and Zanthoxylum chalybeum. The extracts of roots and flowers of Cleome gynandra; root barks, stem barks and leaves of Zanthoxylum chalybeum were used for investigation. The powdered plant materials were macerated in petroleum ether, ethyl acetate, nbutanol and aqueous methanol, then concentrated in vacuum using rotary evaporator. The antimicrobial activity of extracts was evaluated using disc diffusion and macrodilution methods. Phytochemical screening was done by TLC and Chemical test. The cytotoxicity of the extracts was determined by brine shrimp assay. The most extracts contain alkaloids, anthraquinones, flavonoids, phenols, saponin, steroids, tannins and terpenoids, which cause bioactivities against tested species. The least MIC of 0.3125 mg/mL was shown by nBECGR against Staphylococcus aureus with TA (67.2 mL/g), IZ (7.93 ± 0.15) mm AI (0.22), followed by AMECGR with MIC of 0.625 mg/mL, TA of 89.28 mL/g against E. coli, nBEZCL with MIC of 0.625 mg/mL, TA of 36.8 mL/g against Escherichia coli, nBEZCS with MIC of 0.625 mg/mL and TA of 3.04 mL/g against Staphylococcus aureus. The rest extracts show the range of 1.25-10 mg/mL. The nBECGR have highest cytotoxicity with LC50 (18.01 ± 0.38) μg/mL but less than Cyclophosphamide with LC50 of 16.3 μg/mL (Moshi et al., 2010) and nontoxic was shown by PETECGF with LC50 (160.95 ± 2.16) μg/mL. Antimicrobial activities of root extracts of Cleome gynandra and their cytotoxicity are reported for the first time. The observed antimicrobial activity and cytotoxicity of the extracts indicated that the compounds of these species may possess higher antimicrobial efficacy. Therefore, further phytochemical and antimicrobial investigation of isolated compounds is required, and determination of acute toxicity of the extracts and compounds is important.