Purpose This cross-sectional study aimed to estimate the prevalence of Chlamydia trachomatis (CT) infection alone and in combination with human papillomavirus (HPV). Furthermore, the study investigates whether the CT infection increases the risk of contracting HPV and whether the presence of both pathogens is associated with a higher prevalence of cervical lesions. Methods Cervical samples of 1,134 asymptomatic women enrolled in a screening program for cervical cancer were analyzed. Two cervical specimens were collected from each patient, one for cytologic examination and the other for detection of CT by polymerase chain reaction (PCR), using a primer pair which amplifies a specific sequence of the DNA plasmid. Results The overall prevalence rate infection was 10.9 %, being 10 % in the women with normal cytology, 13.8 % in those with atypical squamous cells of undetermined significance (ASC-US), and 25 % with low-grade squamous intraepithelial lesion (LSIL). The infection by CT did not increase the risk of acquiring HPV infection. The higher prevalence of LSIL in women co-infected with HPV and CT is possibly due to HPV. Conclusion CT infection was more prevalent in younger women aged up to 32 years, who had an early onset of reproductive activity and a history of having had multiple sexual partners lifelong may be at a greater risk of acquiring infection of the genital tract by C. trachomatis.