This study evaluated the anticancer, antimicrobial, and hemolytic potential of zinc oxide nanoparticles (ZnO-NPs) synthesized from a novel medicinal plant Lagerstroemia indica. The formation of synthesized ZnO-NPs was confirmed by ultraviolet-visible (UV-Vis) spectroscopy at a peak of 302 nm, and the X-ray diffraction (XRD) analysis confirmed particle size of 19.66 nm. The transmission electron microscopy (TEM) and scanning electron microscope (SEM) with energy dispersive spectroscopy (EDS) micrograph confirmed hexagonal shapes with purity of ZnO. Fourier transform infrared (FTIR) analysis confirmed the presence of various functional groups. The minimum inhibitory concentration (MIC) of ZnO against the four bacteria Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, and Klebsiella pneumoniae was found to be 88 mu g/ml, 52 mu g/ml, 79 mu g/ml, and 72 mu g/ml, respectively, that completely inhibited the bacterial growth. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) test showed significant cytotoxic potential against MCF-7 and HeLa cells with the IC50 concentrations at 36.28 mu g/ml and 30.10 mu g/ml, respectively. ZnO-NPs showed subsequent reduction in mitochondrial membrane potential (MMP) and increased level of reactive oxygen species (ROS). The acridine orange/ethidium bromide (AO/EB) staining showed more number of early apoptotic and late apoptotic cells as compared to the standard drug, Camptothecin. Through real-time quantitative polymerase chain reaction (RT-qPCR), the level of Caspase 3 and p53 was upregulated, and these NPs do not have any hemolytic potential on human RBCs in both MCF-7 and HeLa cell lines. Thus, the synthesized ZnO-NPs from the medicinal plant L. indica could be used as an anticancer agent after further in vivo trials.