The nanoparticles have unique and superior properties which make them applicable in almost every field of human life. Among the various methods that exist for the synthesis of nanoparticles, green synthesis is one of the best economic and eco-friendly methods compared with other conventional chemical methods. The nanoparticles synthesized by this method are also free from toxicity properties. This paper describes one such green synthesis method for zinc oxide nanoparticles (ZnO NPs) using the aqueous flower extract of Senna auriculata. The synthesized nanoparticles (SA-ZnO NPs) have been examined using UV-Vis spectrophotometer, photoluminescence spectroscopy, Fourier-transform infrared spectroscopy (FTIR), transmission electron microscopy (TEM), scanning electron microscopy (SEM), and X-ray diffraction (XRD) analysis. The antibacterial and antifungal activities of the synthesized NPs were evaluated against gram-positive bacteria (S. aureus, B. subtilis), gram-negative bacteria (E. coli, S. typhi) and fungal organisms (C. albicans, A. nigar) using disc diffusion method. Furthermore, the antidiabetic and anticancer activities of the NPs were also been examined by alpha-amylase inhibition assay and MTT assay, respectively. These studies ensured that the SA-ZnO NPs have significant antimicrobial, antidiabetic and anticancer activities.