Dientamoeba fragilis is an inhabitant of human gastrointestinal tract with a worldwide distribution. The first description considered this protozoan a rare and harmless commensal, since then it has struggled to gain recognition as a pathogen. Commercial multiplex real-time PCR was used to detect D. fragilis in fecal samples from hospitalized children (⩽18 years) with acute gastrointestinal disease, admitted to two hospitals of Lisbon area, with different demographic characteristics. A total of 176 children were studied, 103 (58·5%) male, 144 (81·8%) children between 0 and 5 years and 32 (18·2%) above 6 years old. The overall protozoa frequency considering the four tested microorganisms were 8·5% (15/176), and the most frequently found protozoan was D. fragilis, 6·3% (11/176). Dientamoeba fragilis frequency was higher among older children (21·9%), than younger children (2·8%), and greater in boys (6·8%) than in girls (5·5%). All positive children presented with diarrhoea associated with vomiting, fever and abdominal pain. Infection was associated with the age of children (P < 0·001), school attendance (P = 0·002) and consumption of certain foods (P = 0·014), e.g. cakes with crème and ham. The frequency of diantamoebiasis found in a cohort of hospitalized Portuguese children, with acute gastrointestinal disease, could be considered a very high value when compared with the protozoan frequency normally associated with this pathology.