Abstract Background Giardia duodenalis is a widespread parasite of mammalian species, including humans. The prevalence of this parasite in children residing in Portugal is currently unknown. This study intended to estimate G. duodenalis infection prevalence and identify possible associated risk factors in a healthy paediatric population living in the District of the Portuguese capital, Lisbon. Methods Between February 2002 and October 2008, 844 children were randomly selected at healthcare centres while attending the national vaccination program. A stool sample and a questionnaire with socio-demographic data were collected from each child. Giardia infection was diagnosed by direct examination of stools and antigen detection by ELISA. Results The population studied revealed a gender distribution of 52.8% male and 47.2% female. Age distribution was 47.4% between 0-5 years and 52.6% between 6-15 years. The prevalence of Giardia infection was 1.9% (16/844) when estimated by direct examination and increased to 6.8% (57/844) when ELISA results were added. The prevalence was higher among children aged 0-5 years (7.8%), than among older children (5.8%), and was similar among genders (6.9% in boys and 6.5% in girls). The following population-variables were shown to be associated risk factors for G. duodenalis infection: mother's educational level (odds ratio (OR)= 4.49; confidence interval (CI): 1.20-16.84), father's educational level (OR = 12.26; CI: 4.08-36.82), presence of Helicobacter pylori infection (OR = 1.82; CI: 1.05-3.15), living in houses with own drainage system (OR = 0.10; CI: 0.02-0.64) and reported household pet contact, especially with dogs (OR = 0.53; CI: 0.31-0.93). Conclusion The prevalence of giardiasis in asymptomatic children residing in the region of Lisbon is high. Several risk factors were associated with Giardia prevalence and highlight the importance of parents' education and sanitation conditions in the children's well being. The association between G. duodenalis and H. pylori seems an important issue deserving further investigation in order to promote prevention or treatment strategies.