Rare systemic studies concerning prevalence of intestinal parasites in children have been conducted in the second smallest country in Africa, the Democratic Republic of São Tomé and Príncipe. Fecal specimens from 348 children (214 in-hospital attending the Aires de Menezes Hospital and 134 from Agostinho Neto village) in São Tome Island were studied by parasitological and molecular methods. Of the 134 children from Agostinho Neto, 52.2% presented intestinal parasites. 32.1% and 20.2% of these children had monoparasitism and polyparasitism, respectively. Ascaris lumbricoides (27.6%), G. duodenalis (7.5%), T. trichiura (4.5%) and Entamoeba coli (10.5%) were the more frequent species identified in the children of this village. Giardia duodenalis (7.5%) and E. bieneusi (5.2%) were identified by PCR. Nested-PCR targeting G. duodenalis TPI identified Assemblage A (60%) and Assemblage B (40%). The E. bieneusi ITS-based sequence identified genotypes K (57.1%), KIN1 (28.6%) and KIN3 (14.3%). Among the 214 in-hospital children, 29.4% presented intestinal parasites. In 22.4% and 7.0% of the parasitized children, respectively, one or more species were concurrently detected. By microscopy, A. lumbricoides (10.3%) and Trichiuris trichiura (6.5%) were the most prevalent species among these children, and Cryptosporidium was detected by PCR in 8.9% of children. GP60 locus analysis identified 6.5% of C. hominis (subtypes IaA27R3 [35.7%], IaA23R3 [14.3%], IeA11G3T3 [28.6%] and IeA11G3T3R1 [21.4%]) and 2.3% of C. parvum (subtypes IIaA16G2R1 [20.0%], IIaA15G2R1 [20.0%], IIdA26G1 [40.0%] and IIdA21G1a [20.0%]). G. duodenalis and E. bieneusi were identified in 0.5% and 8.9% of the in-hospital children, respectively. G. duodenalis Assemblage B was characterized. The E. bieneusi genotypes K (52.6%), D (26.4%), A (10.5%) and KIN1 (10.5%) were identified. Although further studies are required to clarify the epidemiology of these infectious diseases in this endemic region the significance of the present results highlights that it is crucial to strength surveillance on intestinal pathogens.