Introduction: We evaluated if ascitic fluid sample could be used for the diagnosis of tuberculosis intestinal obstruction (TBIO) by a nested polymerase chain reaction (PCR) assay and analyzed the clinical and laboratory findings in 33 patients with confirmed intestinal tuberculosis (TB) compared to 54 patients with other pathologies of intestinal obstruction. Methods: We tested ascitic fluid sample by Ziehl-Neelson staining; Lowenstein-Jensen (L-J) culture method was used for the culture and PCR was performed for targeting the IS6110 sequence. Results: We found that 87.9% (29/33) of cases with intestinal TB and 0% (0/54) with other causes who had positive results with the nested PCR assay. PCR, in principle, is a highly sensitive technique that detects DNA from a single to a few microorganisms with the overall sensitivity, specificity, and positive predictive value (PPV) of 87.87%, 100%, and 100%, respectively. Acid-fast bacillus (AFB) staining alone has positivity of only 24% and histology alone has positivity of 88%. Conclusion: PCR has a potentially important role in improving the diagnostic accuracy.