Herein, we report a strategy to synthesize zinc oxide nanoparticles (ZnO NPs) from Clitoria ternatea flower extract through green synthesis platform. The bioactive components in the C. ternatea flower extract acts as a stabilizing and capping agent, which enables the synthesis of ZnO NPs without the use of chemical reagents. Synthesis of of ZnO NPs through green platform was confirmed by analytical characterization techniques such as UV–Vis spectrophotometry, XRD, FTIR, DLS, Zeta potential, TEM, and EDX methods. The surface morphology and average size of ZnO NPs investigated through TEM and DLS results indicates rod shaped structure with average particle size of 84 nm. Green synthesized ZnO NPs shows excellent antibacterial, antibiofilm, and antioxidant activity which is dose-dependent. These ZnO NPs have cytotoxicity against breast cancer cells (MCF-7). The MTT test revealed a concentration-dependent inhibitory effect on MCF-7 cells, with IC 50 being 12.1 g/ml, and their anticancer potential was detected utilizing live and dead cell assays LDH release, Ao/EtBr), DAPI, ROS, and caspase-3 activation assays. Finally, the findings of these results shows that ZnO NPs derived from C. ternatea flower extract are highly potential candidates to treat bacterial infection and human breast cancer.