The study’s objective was to quantify the association of sexual orientation with substance use among Colombian adults experiencing homelessness. A secondary analysis of the Census of Street Inhabitants - CHC-2020 was completed. Three thousand five hundred forty-seven participants between 18 and 75 years participated, and 3.16% reported lesbian, gay, or bisexual (LGB) sexual orientation. LGB sexual orientation was associated with alcohol consumption (OR = 2.14, 95%CI 1.46 - 3.14]), cigarettes (OR = 1.70, 95%CI 1.12 - 2.56), cannabis (OR = 1.58, 95%CI 1.07 - 2.33), cocaine (OR = 2.13, 95%CI 1.30 - 3.50) and benzodiazepines (OR = 2.15, 95%CI 1.19-3.89). In conclusion, Colombian adults experiencing homelessness with gay or lesbian sexual orientation have twice the risk of consuming alcohol, cigarettes, cannabis, cocaine, and benzodiazepines than heterosexuals. More studies are needed on this population.