Chlamydia trachomatis (CT), an obligate intracellular bacterium, requires living cells to replicate. Half of men infected with CT are asymptomatic. CT infection can persist for up to four years within couples and affect their fertility. Chlamydia infection in men acts as a reservoir for transmission to women and can cause urinary tract inflammation, sperm DNA damage, and acute epididymitis. Semen samples from 1080 subfertile patients with normal and abnormal spermograms were examined to detect the presence of CT. An ELISA test was used to detect the presence of anti-CT IgA in these patients' seminal plasma. CT infection was also confirmed by molecular investigation using specific primers. In order to evaluate the effect of CT infections on the DNA Fragmentation Index (DFI), 40 CT-infected cases and 20 CT-negative controls were analyzed by a Sperm Chromatin Structure Assay using flow cytometry. Among 1080 patients with poor sperm parameters, 155 (14.3%) patients were diagnosed with CT, 11% among those with semen abnormalities and 26% among those without semen abnormalities patients. The DFI was statistically higher in cases than in controls (p < 0.05). Given the prevalence of infection and also the high frequency of asymptomatic CT infection among infertile individuals with poor sperm parameters, screening for infection in these patients is essential in order to avoid adverse sequelae. We propose that the higher rate of DFI in CT-infected infertile men might be an underlying cause of their infertility and this warrants greater attention.