Acacia nilotica is used traditionally to manage several diseases including Diabetes mellitus, however, its efficacy and safety is not well evaluated. The aim of this study was to determine in vivo the hypoglycemic activity and safety of the aqueous stem bark extracts of this plant in male swiss white albino mice. The anti-diabetic activity was screened in alloxan induced diabetic mice using oral and intra-peritoneal routes. The safety of this plant extract was studied in mice that were orally and intraperitoneally administered with 1 g/kg body weight daily for 28 days by recording changes in body and organ weight, hematological and biochemical parameters and histology. Mineral composition was estimated using total reflection X-ray fluorescence system and atomic absorption spectrometry. Phytochemical composition was assessed using standard procedures. The extract administered at 50, 100, 200, 300 mg/kg body weight showed hypoglycemic activity. The Intraperitoneal route was more effective compared to the oral route. Intraperitoneal administration of the extract at 1 g/kg body weight significantly reduced body weight gain, percent organ to body weight of testes, while oral administration at the same dose decreased levels of platelets. Oral administration of the aqueous stem bark extracts of A. nilotica at 1 g/kg body weight caused increase in levels of γ-glutamyl transpeptidase, Creatine kinase, and Total bilirubin while decreasing levels of alanine transaminase, aspartate aminotransferase, α-Amylase, and Alkaline phosphatase. Intraperitoneal administration of the same dose decreased levels of aspartate aminotransferase. The aqueous stem bark extract of A. nilotica contained tannins, total phenols, flavonoids, saponins, and alkaloids. Sodium, chloride, potassium, calcium, titanium, vanadium, chromium, manganese, iron, copper, zinc, arsenic, nickel, lead, and cadmium were present in the aqueous stem bark extracts of A. nilotica at levels below the recommended daily allowance. In conclusion, the observed hypoglycemic activity and slight toxicity could be associated with the phytochemicals present in this plant extract.