BACKGROUND: Acute anal fissure as a common disease in society has several etiologies and manifestations such as severe anal pain and bleeding. Nitroglycerin ointment 0.2% is the most common topical treatment used. The most common side effect of nitroglycerin is headache, which is annoying for patients and often leads to discontinuation of the drug. OBJECTIVE: Comparison of egg yolk as a natural substance with analgesic and anti-inflammatory properties and minimal side effects with nitroglycerin ointment in the treatment of acute anal fissure. METHODS: This randomized clinical trial was carried out during a 10-day period in the Gastroenterology clinic of Ghaem Hospital, Mashhad, Iran (year 2015). 126 patients who filled the inclusion criteria were enrolled. The patients were randomly divided into two groups. Nitroglycerin ointment (0.2%) was applied by patients in the first group, twice daily for 10 days. For the second group, one egg yolk once a day was administered rectally up to 10 days. The pain and bleeding severity were recorded every two days up to 10 days after finishing the treatment course, based on visual scale. RESULTS: The results showed that egg yolk caused a significant reduction in pain and bleeding compared with nitroglycerin (p<0.05). At the beginning of the study, the difference in pain intensity between the two groups was not statistically significant (p-value = 0.25). However, it became significant in the following days. Changes in the frequency of rectorrhagia were also significant in both groups, showing a major decrease in the number of rectorrhagia cases (p<0.001). CONCLUSION: Egg yolk is more efficient than nitroglycerin in the treatment of acute anal fissure. In addition, lack of any side effects results in the completion of the treatment course by the patients. TRIAL REGISTRATION: The trial was registered at the Iranian Registry of Clinical Trials (http://www.irct.ir) with the Irct ID: IRCT2015050718915N3. FUNDING: This work was supported by a grant from the Vice Chancellor of Research of Mashhad University of Medical Sciences.