Juglans regia L., commonly known as walnut, is a valuable medicinal herb with the potency to treat various conditions and illnesses including diabetes, rheumatism, fever and skin illnesses. This study assessed the acute toxicity, anti-diabetic and anti-atherogenic activities of the hydro-alcoholic extract of the walnut root bark of Juglans regia L. (Juglandaceae), in vivo, using Wistar rats. The toxicological effects of the hydro-alcoholic extract of walnut bark Juglans regia L: are still elusive. In order to evaluate the toxicity profile of this plant, rats were orally treated with a single concentration of 2000 mg/kg and observed during a period of two weeks. For the anti-diabetic study, thirty male wistar rats (130–170 g) were randomized into 5 groups (n = 6/group). Groups I and II served as negative and normal controls, respectively. Diabetes was induced in test groups (II–V) using 200 mg/kg of body weight (BW) streptozotocin. Concerning the clincial outcomes, no mortality, morbidity, or abnormal hematological, biochemical and histopathological alterations were observed. Accordingly, J. regia L is considered a non-toxic plant. The extract was found to limit weight loss and reduce blood glucose levels by −32.30% after 14 days of treatment for the anti-diabetic and anti-atherogenic study. The extract also reduced dyslipidaemia. Our data indicated that J. regia L contains bio-compounds that may alleviate chronic hyperglycemia while preventing cardiovascular complications by improving dyslipidaemia. This could be a potential herb for future studies to develop more effective drugs for improving glycemic and cholesterol control.