Jejunal fluid culture is the gold standard for assessing jejunal microflora. Aspiration of jejunal fluid is sometime difficult. As the microorganisms rests on the mucosal surface, culture of the mucosal biopsy may be a possible alternative method. To study the role of jejunal mucosal biopsy culture to assess jejunal microflora and to compare it with jejunal fluid culture. Thirty adult subjects with gastroesophageal reflux disease requiring endoscopy underwent enteroscopy. Jejunal fluid aspirate and mucosal biopsy were cultured. The procedure was repeated after omeprazole therapy in 18 patients. Forty-eight pairs (30 preomeprazole therapy and 18 postomeprazole therapy) of fluid and mucosal biopsies were cultured. In 45 of the 48 pairs (94%), both the culture of jejunal biopsy and jejunal fluid yielded similar results with respect to the presence (n = 27) or absence of growth (n = 18). In the remaining 3 pairs, the growth was present either in the biopsy culture (n = 2) or in the fluid culture (n = 1) only. Among those pairs in which growth was present, microorganisms isolated were identical in 53%, differed by ≤ 2 organism in 37% and different by >2 organisms in 10%. Ten of the 12 patients who were detected to have small intestinal bacterial overgrowth (SIBO) on fluid culture were also detected to have SIBO on biopsy culture. Sensitivity, specificity, positive and negative predictive value of biopsy culture in diagnosing SIBO was 83.5%, 97.2%, 94.7%, and 91.6%, respectively. Culture of unwashed endoscopic jejunal mucosal biopsy is an effective and simple alternative to jejunal fluid culture for assessing jejunal microflora.