Infections by the protozoan enteroparasites Giardia duodenalis and Cryptosporidium spp are a major cause of morbidity in children attending day care facilities in developed countries. In this cross-sectional study, we aimed to estimate the occurrence and genotype frequencies of these pathogens in children attending day care centers in Majadahonda, Central Spain. To do so, single stool samples were obtained from 90 children and tested for the presence of G duodenalis and Cryptosporidium spp by conventional microscopy and immunochromatography. Positive results by these techniques were subsequently confirmed by immunofluorescence microscopy. G duodenalis-positive samples were subjected to molecular characterization studies by multilocus sequence-based genotyping of the glutamate dehydrogenase and β-giardin genes of the parasite. G duodenalis assemblages were confirmed by restriction fragment length polymorphism analyses and sequencing. A socioepidemiological questionnaire was used to identify variables potentially associated with giardiasis/cryptosporidiosis in the population of children under investigation. Overall, G duodenalis and Cryptosporidium spp were detected in 15.5% and 3.3% of stool samples, respectively. Giardiasis and cryptosporidiosis were found in 3/3 and 2/3 day care centers, respectively, affecting mainly infants aged 13 to 24 months. A total of 8 G duodenalis isolates were confirmed as subassemblage BIV, all of them belonging to asymptomatic children. Attempts to genotype Cryptosporidium isolates failed. None of the variables considered could be associated with higher risk of infection with giardiasis or cryptosporidiosis. These results clearly indicate that asymptomatic infections with G duodenalis and Cryptosporidium spp are frequent in <3-year-old children in Central Spain.