The current study was designed to investigate the beneficial role of diosmin, a biologically active flavonoid, against methotrexate‐ (MTX‐) induced hepatic, renal, and cardiac injuries in mice. Male Swiss albino mice received a single intraperitoneal injection of MTX (at 20 mg/kg, body weight) either alone or in combination with oral diosmin (at 50 or 100 mg/kg body weight, for 10 days). Serum was used to evaluate tissue injury markers, while hepatic, renal, and cardiac tissue samples were obtained for determination of antioxidant activity as well as histopathological examination. Diosmin treatment ameliorated the MTX‐induced elevation of serum alkaline phosphatase, aminotransferases, urea, creatinine, lactate dehydrogenase, and creatine kinases as well as plasma proinflammatory cytokines (interleukin‐1‐beta, interleukin‐6, and tumor necrosis factor‐alpha). Additionally, both diosmin doses significantly reduced tissue levels of malondialdehyde and nitric oxide and increased those of glutathione, glutathione peroxidase, glutathione reductase, glutathione S‐transferase, superoxide dismutase, and catalase, compared to the MTX‐intoxicated group. Histopathological examination showed that diosmin significantly minimized the MTX‐induced histological alterations and nearly restored the normal architecture of hepatic, renal, and cardiac tissues. Based on these findings, diosmin may be a promising agent for protection against MTX‐induced cytotoxicity in patients with cancer and autoimmune diseases.