The anti-cancer, anti-aging, anti-inflammatory, antioxidant, and anti-diabetic effects of zinc oxide nanoparticles (ZnO-NPs) produced from aqueous leaf extract of Aquilegia pubiflora were evaluated in this study. Several methods were used to characterize ZnO-NPs, including SEM, FTIR, XRD, DLS, PL, Raman, and HPLC. The nanoparticles that had a size of 34.23 nm as well as a strong aqueous dispersion potential were highly pure, spherical or elliptical in form, and had a mean size of 34.23 nm. According to FTIR and HPLC studies, the flavonoids and hydroxycinnamic acid derivatives were successfully capped. Synthesized ZnO-NPs in water have a zeta potential of -18.4 mV, showing that they are stable solutions. The ZnO-NPs proved to be highly toxic for the HepG2 cell line and showed a reduced cell viability of 23.68 ± 2.1 % after 24 hours of ZnO-NP treatment. ZnO-NPs also showed excellent inhibitory potential against the enzymes acetylcholinesterase (IC50: 102 μg/mL) and butyrylcholinesterase (IC50: 125 μg/mL) which are involved in Alzheimer’s disease. Overall, the enzymes involved in aging, diabetes, and inflammation showed a moderate inhibitory response to ZnO-NPs. Given these findings, these biosynthesized ZnO-NPs could be a good option for the cure of deadly diseases such as cancer, diabetes, Alzheimer’s, and other inflammatory diseases due to their strong anticancer potential and efficient antioxidant properties.