Dientamoeba fragilis is a pathogenic protozoan parasite that is implicated as a cause of human diarrhoea. A case-controlled study was conducted to determine the clinical signs associated with D. fragilis infection in children presenting to a Sydney Hospital. Treatment options are also discussed. Stool specimens were collected from children aged 15 years or younger and analysed for the presence of D. fragilis. In total, 41 children were included in the study along with a control group. Laboratory diagnosis was performed by microscopy of permanently stained, fixed faecal smears and by real-time PCR. Gastrointestinal symptoms were present in 40/41 (98%) of these children with dientamoebiasis, with diarrhoea (71%) and abdominal pain (29%) the most common clinical signs. Chronic gastrointestinal symptoms were present in 2% of cases. The most common anti-microbial used for treatment was metronidazole (n=41), with complete resolution of symptoms and clearance of parasite occurring in 85% of cases. A treatment failure rate occurred in 15% of those treated with metronidazole. Follow-up treatment comprised of an additional course of metronidazole or iodoquinol was needed in order to achieve complete resolution of infection and symptoms in this group. This study demonstrates the pathogenic potential of D. fragilis in children and as such it is recommended that all laboratories must routinely test for this organism and treat if detected.