Human papillomavirus (HPV) and Chlamydia trachomatis are pathogens with oncogenic potential associated with persistent infections. Epidemiological data on C. trachomatis infection status, C. trachomatis/HPV co-infection and the relationship between HPV genotypes in Italian women are only preliminary. The aim of the present study was to characterize the relationship between HPV genotypes and C. trachomatis in an extending cohort of asymptomatic immunocompetent women from an area of north-east Italy. A retrospective study was conducted using Luminex technology on cervical swabs from asymptomatic immunocompetent women, comprising 921 attending the prevention centre for the Cervical Cancer Program and 6214 who had been referred to the Sexually Transmitted Infections Center, with clinical indications of HPV and C. trachomatis infections. A quantitative real-time PCR was performed to assess chronic C. trachomatis infection by heat-shock protein 60 (Hsp60) gene expression. The overall prevalence of the investigated pathogens was 39 % (359/921) for HPV and 4 % (251/6214) for C. trachomatis. The Hsp60 gene was detected in 57 % of the women infected with C. trachomatis. HPV co-infection was present in 58 % of C. trachomatis-infected women. A high prevalence of co-infection was found in women with chronic C. trachomatis infection (68 %, P = 0.0002), especially in women ≤ 25 years (72 %) where HPV multiple infections were found in 78 % (P = 0.022). HPV genotype distribution showed that uncommon low-risk genotypes were associated with C. trachomatis. These results indicate a high frequency of co-detection of multiple HPV genotypes in chronically infected young women and suggest that the expression of the C. trachomatis Hsp60 gene may favour HPV infection.