The clinical significance of one of the most prevalent single-celled intestinal parasites worldwide, Blastocystis, remains unsettled. A plethora of clinical and epidemiological studies have been undertaken to generate data on its prevalence in different populations and investigate the role of the parasite as a cause of gastro- and extra-intestinal disease. In this article, we pinpoint limitations of studies that seek to determine the clinical significance of Blastocystis, based on shortcomings in our understanding of Blastocystis diagnosis and biology, and identify methodologies for further studies aimed at determining the molecular epidemiology and clinical impact of this parasite.