The increasing use of medicinal plants in traditional and modern therapies necessitates thorough toxicological evaluations to ensure their safety. Chrozophora genus includes 7-8 species that belong to the Euphorbiaceae family, distributed in Pakistan, India, West Africa, and the Mediterranean with four species found in the Kingdom of Saudi Arabia (KSA). This study evaluates the subacute toxicity of the ethyl acetate extract (EtOAcE) of Chrozophora tinctoria in male and female rats. Over 14 days, rats were daily administered the extract at doses of 50, 100, and 200 mg. Mortality was observed at a rate of one rat per group. Female rats showed a significant reduction in body weight, while organ weights remained unaffected. Kidney function tests revealed significant increases in bilirubin and creatinine levels in male rats at 200 mg, and uric acid levels in female rats at the same dosage. Liver enzyme analysis indicated significant elevations in ALP, ALT, and GGT levels in both sexes at various dosages. Glucose levels increased significantly in female rats at 200 mg/kg, with no significant changes in cortisol levels in either sex. Histopathological examinations demonstrated notable abnormalities in liver, kidney, spleen, and small intestine tissues. These data demonstrate the essentiality of careful evaluation and monitoring when considering C. tinctoria for medicinal use, highlighting the need for further studies on its long-term safety and therapeutic index.