Objective To determine the prevalence of Chlamydia trachomatis (CT) and Neisseria gonorrhoeae (NG) in the homeless population in Medellín, Colombia, using molecular diagnostic methods. It also intended to develop a demographic profile, exploring associated factors and the dynamics of the social and sexual interactions of this community. Design Cross-sectional study. Setting Two homeless care centres in Medellín, Colombia. Participants Homeless individuals that assisted to the main homeless care centres of Medellín, Colombia from 2017 to 2019. Primary and secondary outcome measures The prevalence of CT and NG in this population using qPCR detection, factors associated with CT and NG infection, and the sociodemographic profile of the community. Results The prevalence of CT infection was 19.2%, while that of NG was 22.6%. Furthermore, being a female was significantly correlated to CT infection p<0.05 (adjusted OR, AOR 2.42, 95% CI 1.31 to 4.47). NG infection was significantly associated with factors such as: sexual intercourse while having a sexually transmitted infection p<0.05 (AOR 3.19, 95% CI 1.48 to 6.85), having more than 11 sexual partners in the last 6 months p=0.04 (AOR 2.91, 95% CI 1.04 to 8.09) and having daily intercourse p=0.05 (AOR 3.15, 95% CI 1.02 to 9.74). Conclusions The prevalence of CT and NG was higher than that reported in the general population. Additionally, females had a higher percentage of infection compared with males.