Dientamoeba fragilis is a pathogenic trichomonad found in the gastrointestinal tract of humans and is implicated as a cause of diarrhoea. Despite its discovery over a century ago, there has been no recent thorough description of this parasite by microscopy. Scanning electron microscopy, transmission electron microscopy, confocal and light microscopy were therefore used to characterise D. fragilis populations growing in xenic culture. Two different populations - smooth and ruffled cells - were identifiable by scanning electron microscopy. No flagella, pelta structures, undulating membrane or pseudocyst-like forms were present. The organelles in D. fragilis were analysed by transmission electron microscopy; like Trichomonas and Histomonas, D. fragilis contains hydrogenosomes that presumably represent the site of anaerobic respiration. The nuclear morphology of D. fragilis trophozoites grown in vitro and trophozoites from clinical isolates were also compared by confocal microscopy and light microscopy. The majority of cells grown in culture were mononucleate while most cells in permanent stained faecal smears were binucleate. The two nuclei of D. fragilis are morphologically indistinguishable and contain equivalent amounts of DNA as determined by DAPI staining. The approximate cell and nuclear volume of four isolates of D. fragilis were measured and shown to be comparable to other trichomonads. In addition, the discovery of a virus-like particle is reported, to our knowledge for the first time in D. fragilis. This study therefore provides extensive and novel details of the ultrastructure of a neglected protozoan parasite that is an emerging cause of human disease.