Introduction: There are no reports of the potential sources of Blastocystis sp. in Colombia.Objectives: To estimate the frequency of Blastocystis sp. and explore possible associations among infection, clinical manifestations and environmental sources.Materials and methods: A descriptive study was conducted; a representative sample was calculated from a population of 1,993 children less than 5 years old of the lowest socioeconomic levels (1 and 2) attending day care centers in Calarcá, Colombia. The calculated sample size was 275 children, who were selected randomly in 10 % of the centers. Stool testing, questionnaires and sampling to identify the parasite in feces, nails, pets and environmental sources, were performed. Fresh preparations were made with 1% iodine solution, and a concentration technique with formalin-ether was used. Prevalence and odd ratios were estimated; the chi-square test was utilized for qualitative variables, and Student´s t for the quantitative ones.Results: The prevalence of infection in children was of 57.5%. A statistically significant association was found between the Blastocystis sp. positive stool tests and the presence of the parasite in containers of sugar cane beverage (OR=1.70; 95% CI: 1.05-2.79), wood or ground floors (OR=3.43; 95% CI: 1.12-10.48) and in foods other than bottled milk (OR=3.23; 95% CI: 1.41-4.41). A non-significant high prevalence was found in nails (OR=1.62; 95% CI: 0.99-2.67), as well as a significantly higher prevalence of diarrhea (OR=1.95; 95% CI: 0.99-2.77) in children with Blastocystis sp. (45%) as compared with those without the parasite (33%; p<0.05).Conclusions: Blastocystis sp. showed a significant association with diarrhea symptoms in children 0 to 5 years old and was widely distributed in the environment and in water. It is necessary to determine if there are specific hosts with pathogenic potential through molecular techniques.