Renal calculi formation is one of the most common urological disorders. Urinary stone disease is a common disease, which affects 10‑12% of the population in industrialized countries. In males, the highest prevalence of the disease occurs between the age of 20 and 40 years, while in females, the highest incidence of the disease occurs later. Previous studies have shown that long‑term exposure to oxalate is toxic to renal epithelial cells and results in oxidative stress. In the present study, a methanolic extract of aerial parts of Urtica dioica was screened for antiurolithiatic activity against ethylene glycol and ammonium chloride‑induced calcium oxalate renal stones in male rats. In the control rats, ethylene glycol and ammonium chloride administration was observed to cause an increase in urinary calcium, oxalate and creatinine levels, as well as an increase in renal calcium and oxalate deposition. Histopathological observations revealed calcium oxalate microcrystal deposits in the kidney sections of the rats treated with ethylene glycol and ammonium chloride, indicating the induction of lithiasis. In the test rats, treatment with the methanolic extract of Urtica dioica was found to decrease the elevated levels of urinary calcium, oxalate and creatinine, and significantly decrease the renal deposition of calcium and oxalate. Furthermore, renal histological observations revealed a significant reduction in calcium oxalate crystal deposition in the test rats. Phytochemical analysis of the Urtica dioica extract was also performed using liquid chromatography‑electrospray ionization tandem mass spectrometry and high-performance liquid chromatography with photodiode array detection, to determine the chemical composition of the extract. The eight chemical constituents identified in the extract were protocatechuic acid, salicylic acid, luteolin, gossypetin, rutin, kaempferol‑3‑O‑rutinoside, kaempferol‑3‑O‑glucoside and chlorogenic acid. In conclusion, the results of the present study suggest that Urtica dioica has strong antiurolithiatic activity and may have potential as a natural therapeutic agent for various urological disorders.