Giardia duodenalis is a common gastrointestinal pathogen globally that has been associated with growth failure in children. Most of the studies have been done in school-age children, and there is a paucity of data in pre-school children. We determined the prevalence and factors associated with G. duodenalis infection in children aged 9-36 months presenting to Mulago Hospital with diarrhea or cough. Demographic and socio-economic characteristics, animal ownership, medical history, and physical examination findings were recorded. Stool was tested for G. duodenalis using real-time quantitative polymerase chain reaction (qPCR), and additional tests included stool microscopy and qPCR for Cryptosporidium. The overall prevalence of G. duodenalis infection was 6.7% (214/3,173). In children with diarrhea the prevalence was 6.9% (133/1,923), whereas it was 6.5% (81/1,250) in those with cough as the main symptom. Of 214 children with G. duodenalis infection, 19 (8.9%) were co-infected with Cryptosporidium. Older children (25-36 months) were more likely to have G. duodenalis infection (adjusted odds ratio [aOR]: 2.93, 95% CI: 1.93-4.43). Use of an unimproved toilet (aOR: 1.38, 95% CI: 1.04-1.83) and the wet season (aOR: 1.33, 95% CI: 1.00-1.77) were associated with increased infection. Other factors associated with infection were recurrent diarrhea (aOR: 2.46, 95% CI: 1.64-3.70) and passing of mucoid stool (aOR: 2.25, 95% CI: 1.08-4.66). Having a ruminant at the homestead was also associated with infection (aOR: 1.83, 95% CI: 1.20-2.79). Giardia duodenalis infection occurred in 1 of 15 children aged 9-36 months with diarrhea or cough in Kampala, Uganda. Further studies are needed to clarify the zoonotic significance of G. duodenalis infection in this setting.