Herein, zinc oxide nanoparticles (ZnO NPs) were greenly synthesized from Tridax procumbens aqueous leaf extract (TPE) and characterized physically (e.g., Fourier-transform infrared (FTIR) spectroscopy and scanning electron microscopy (SEM)) and biologically (test of their anti-diabetic activity). Anti-diabetic activities of TPE and TPE-derived ZnO NPs have been carried out in a streptozotocin (STZ)—induced diabetic rat model. Diabetes mellitus (DM) was induced with a single intraperitoneal dosage of the glucose analogue STZ (55 mg/Kg) known to be particularly toxic to pancreatic insulin-producing beta-cells. TPE and TPE-derived ZnO NPs were administered orally, once every day for 21 days in diabetic rats, at 100 and 200 mg/Kg, respectively. The standard antidiabetic medication, glibenclamide, was used as a control at a dose of 10 mg/Kg. Various parameters were investigated, including bodyweight (bw) variations, glycemia, lipidaemia, glycated hemoglobin (HbA1c), and histopathological alterations in the rat’s liver and pancreas. The TPE-mediated NPs were small, spherical, stable, and uniform. Compared to TPE and, to a lesser extent, glibenclamide, TPE-derived ZnO NPs lowered blood glucose levels considerably (p < 0.05) and in a dose-dependent manner while preventing body weight loss. Further, positive benefits for both the lipid profile and glycated hemoglobin were also noticed with TPE-derived ZnO NPs. The histopathological assessment revealed that synthesized TPE-derived ZnO NPs are safe, non-toxic, and biocompatible. At 200 mg/Kg/day, TPE-derived ZnO NPs had a more substantial hypoglycemic response than at 100 mg/Kg/day. Thus, in this first reported experimental setting, ZnO NPs biosynthesized from the leaf extract of Tridax procumbens exert more potent anti-diabetic activity than TPE and glibenclamide. We conclude that such a greenly prepared nanomaterial may be a promising alternative or complementary (adjuvant) therapy, at least to the current Indian’s traditional medicine system. Translational findings are prompted in human populations to determine the efficacy of these NPs.