In Germany, an increase of notified giardiasis was observed between 2002 (n = 3,101) and 2007 (n = 3,651) with 62% of cases acquired in Germany. The highest incidence was reported in 1- to 5-year-olds (2001-2007, 11.5/100,000 on average) and Berlin is one of the most affected states (17.5/100,000). We performed a cross-sectional study in five Berlin kindergartens differing in socioeconomic status and migrant proportion in order to estimate the prevalence and investigate routes of transmission among children under 6 years of age. Stool samples were screened for Giardia lamblia and Cryptosporidium parvum using microscopical and antigen detection. Giardia-positive samples underwent PCR and subtyping. Two hundred two children participated (mean age 3.4 years). We found three girls (1.5%) who tested positive for G. lamblia genotype-A3, all clustering in one kindergarten (prevalence 5.5%). No common source was identified. Two children were symptomatic. Possible dog-to-child transmission was established for one of the symptomatic cases. All contact-tracing results were negative. Other microscopically detected parasites (Blastocystis hominis, Endolimax nana cysts, Entamoeba coli cysts and Iodamoeba bütschlii cysts) were found in 4% of children. In summary, a substantial level of Giardiasis may be prevalent in some of Berlin's kindergartens, despite standard hygienic measures being followed. The relatively high prevalence in one kindergarten indicates the need for further studies to identify risk factors for children, which may help to guide possible interventions and strategies. Giardiasis should be considered as a differential diagnosis in children with unclear gastrointestinal symptoms. Additional education and training on proper toilet and food hygiene may further reduce the possibility of child-to-child transmission.