BACKGROUND & AIMS: The parasites Dientamoeba fragilis and Blastocystis have been detected in feces from patients with irritable bowel syndrome (IBS), therefore these parasites may be involved in IBS pathogenesis. We proposed that a higher prevalence of the parasites in IBS subjects compared with asymptomatic controls would support such a mechanism. We aimed to determine the prevalence of these parasites in IBS subjects (cases) and controls and to identify risk factors associated with parasite carriage. METHODS: We performed a population-based, case-control study of an adult population from an internet-based research institute in Denmark. In January 2010, subjects completed a questionnaire based on the Rome III criteria for IBS and answered questions on factors associated with parasite carriage. Respondents (n = 483) were asked to submit fecal samples for parasite testing; samples were analyzed from 124 cases and 204 controls. RESULTS: A greater proportion of controls than cases carried the parasites (50% vs 36%; P = .01). D fragilis was detected in a greater proportion of fecal samples from controls than cases (35% vs 23%; P = .03), as was Blastocystis (22% of controls vs 15% of cases; P = .09), and a greater percentage of controls carried more than 1 species of parasite (16% of controls vs 8% of cases; P = .05). D fragilis infection was associated with having children 5 to 18 years old in the household and Blastocystis infection was associated with high income (≥600,000 Danish Kroner/y, approximately $100,000 US dollars/y), no animals in the household, and drinking bottled water. CONCLUSIONS: D fragilis and Blastocystis were detected in a greater proportion of fecal samples from the asymptomatic background population in Denmark than from subjects with IBS symptoms. These findings indicate that these parasites are not likely to have a direct role in the pathogenesis of IBS. Longitudinal studies are required to understand their role in gastrointestinal health.