For the first time in this study, Zinc oxide nanoparticles were biosynthesized by the eco-friendly and cost-effective procedure using Amygdalus scoparia stem bark extract then used as antibacterial, antifungal, anticancer, and anti-diabetic agents. The characterization techniques confirmed the biosynthesis, crystalline nature, structure, size, elemental composition of ZnO NPs and bioactive compounds that exist in A. scoparia extract accounting for Zn2+ ion reduction, capping and stabilization of ZnO NPs. The ZnO NPs displayed remarkable inhibitory activity against E. coli, E. aerigenes, S. aureus, P. oryzae, F. thapsinum, and F. semitectum compared to antibiotic standards. The ZnO NPs showed significant inhibitory effects on cancer cell lines, while it had no toxic effect on Vero normal cell line. The ZnO NPs (30 mg/kg)-treated diabetic rats showed significantly higher levels of insulin and lower AST, ALT and blood glucose compared with the STZ induced diabetic group and other treated groups (P < 0.05). The ZnO NPs- and extract-treated rats showed significantly higher levels of IR, GluT2, and GCK expression and lower TNFα expression compared with the STZ induced diabetic rats. Our findings showed that ZnO NPs represented an outstanding performance for biological applications.