Diabetes mellitus is a chronic metabolic disorder characterized by elevated blood glucose levels. This research project aimed to investigate the anti-diabetic potential of a Polyherbal Preparation (PHP) composed of dried leaves of Tinospora cordifolia, dried bark of Cinnamomum zylanicum, dried seeds of Trigonella foenum, and dried seeds of Nigella sativa in streptozotocin-induced diabetes in Albino Wistar rats. The individual plants were extracted using n-hexane, petroleum ether, and ethanol, and prepared 15 different PHPs in varying proportions. The PHPs were then evaluated for their in vitro antioxidant activity, and the ethanol extract exhibited superior scavenging activity against DPPH and hydrogen peroxide radicals compared to the other extracts. Further investigations were conducted to assess the antidiabetic potential of the PHPs. In non-diabetic rats, the PHPs exhibited a dose-dependent glucose-reducing effect, with the most significant reduction observed at a dose of 400 mg/kg body weight. Glucose levels gradually and significantly decreased in the PHP-treated groups, reaching near-normal levels by the 28th day. The glucose-lowering effect at the 400 mg/kg dose was comparable to that of the standard antidiabetic drug, glibenclamide. In diabetic rats, treatment with the PHPs led to a significant reduction in glucose levels compared to the diabetic control group. Notably, the ethanol extract of PHP 1 (EPHP 1) demonstrated potent anti-diabetic activity, similar to that of glibenclamide. These findings provide scientific evidence supporting the anti-diabetic potential of the PHPs. EPHP 1 emerged as a promising candidate for further development as a natural alternative for diabetes management.