Background: Endoscopy is commonly performed in patients with chronic human immunodeficiency virus (HIV)-related diarrhea after negative stool studies. The aim of this study was to determine the diagnostic yield and cost-effectiveness of endoscopy in this setting. Methods: Consecutive HIV- infected patients with chronic unexplained diarrhea who were referred for diagnostic endoscopy were identified. Patient charts, pathology reports, and endoscopy records were reviewed. Results: A total of 479 endoscopic procedures were performed in 307 patients. A pathogen was identified in 147 patients (47.9%); cytomegalovirus was the most frequent organism found (21.5%). The average cost of identifying a pathogen by endoscopy was $3822.94. Colonoscopy had a greater diagnostic yield than flexible sigmoidoscopy (38.7% vs. 22.4%, p = 0.009) and was more cost-effective. The yield of upper endoscopy was 29.6%. In patients with a CD4 count of less than 100/mm3, endoscopy had a higher diagnostic yield (62.8% vs. 8.3%, p < 0.0001) and a lower cost of identifying a pathogen ($2943.92 vs. $21,583.51) than in those with higher CD4 counts. Conclusions: Endoscopy frequently identifies a pathogen in HIV-related chronic diarrhea. Colonoscopy is the most cost-effective procedure. Endoscopic evaluation has a significantly higher diagnostic yield and is considerably more cost-effective in patients with a CD4 count of less than 100/mm3 than in those with higher CD4 counts.