Background and Aim: Small intestinal bacterial overgrowth (SIBO) is associated with gastrointestinal pathology and colonoscopy. This endoscopic investigation could cause changes in gut flora including the occurrence of SIBO. We looked in this study for the effect of colonoscopy (preparation and intubation) on the occurrence of SIBO. Materials and Methods: Prospective study including thirty patients with irritable bowel syndrome (IBS) diagnosed according to Rome III criteria. Two groups were designed: Twenty IBS patients that performed colonoscopy (G1) and ten IBS patients (G2) not referred to colonoscopy. All patients have been tested for the presence of SIBO using glucose hydrogen breath tests (GHBT) at the beginning of the study, on day 1. G1 patients have also been tested before colonoscopy (day 2) and 1 week after (day 9). G2 patients performed GHBT on day 1 and on day 9. Results: The peak value of expired H2 was assessed, and the mean value was calculated. There were no significant statistical differences between the mean H2 values in the 2 groups of patients on day 1. The mean level of H2 significantly decreased after preparing for colonoscopy in G1 patients (P < 0.0001). There were no significant statistical differences between the mean levels of H2 on day 2 versus day 9 in G1 patients (P = 0.176). The mean level of H2 1 week after performing colonoscopy (7.65 ppm) is higher than that obtained after preparing for it (6.3 ppm), but no statistical significance. Patients from G2 showed no statistical differences between the mean levels of H2 on day 1 versus day 9 (P = 0.6132). Patients in G1 had a significantly lower mean H2 level versus G2 patients on day 9. Conclusions: Colonoscopy does not produce SIBO. Preparing for colonoscopy influences the level of expired H2, it reduces the number of intestinal bacteria, probably trough a mechanic effect or by inflating air during the procedure. Performing GHBT too soon after colonoscopy might result in false negative results of GHBT.