Background and Objectives: Plant metabolites, like antimicrobial and cytotoxic effects, exhibit therapeutic benefits.The crude extract of Cyperus rotundus (CR) rhizomes demonstrates such activities; however, the specific components responsible for these functions have not been determinedThis study identifies the bioactive alkaloids in CR rhizome extractMethods: The crude methanolic extract of CR rhizomes was acidified, followed by basification.The precipitate was then purified using sequential chromatog. and high-performance liquid chromatog. techniques.Structural elucidation of the derived compound was performed using the Fourier-transform IR spectroscopy, proton NMR, and mass spectroscopy.Disc dilution and diffusion assays were used to evaluate the isolated compound's min. inhibitory concentrations (MICs) and min. bactericidal concentrations (MBCs) against selected microbial species.The cytotoxicity of the isolated compound was determined against HeLa cancer cells using MTT cell proliferation assays.Results: Spectral anal. confirmed the presence of norrotundine-6-O-glucoside plus appreciable amounts of flavonoids nootkatone, quercetin, chlorogenic acid, catechin, and β-sitosterol.The isolated alkaloid inhibited the growth of Staphylococcus aureus (MIC=MBC=64 μg/mL), Escherichia coli (MIC=100 μg/mL), and Candida albicans (MIC=MBC=64 μg/mL).The alkaloid's half-maximal inhibitory concentration (IC50) was 203.7 μg/mL.The obtained alkaloid from the CR rhizomes' crude extract revealed bactericidal and fungicidal effects against S. aureus and C. albicans, resp., at 64 μg/mL, bacteriostatic against E. coli at 100 μg/mL, and cytotoxic against HeLa cells at an IC50 equal to 203.7 μg/mL.Conclusion: The elucidated alkaloid from the CR rhizomes' crude extract exhibited bactericidal and fungicidal effects against S. aureus and C. albicans, resp., at 64 μg/ mL, bacteriostatic activity against E. coli at 100 μg/mL and cytotoxicity against HeLa cells at IC50 of 203.7 μg/mL.