Background/Objectives: Infections of the urogenital tract have experienced renewed interest in recent years, due to their frequency and also their impact on sperm parameters and the fertilizing quality of spermatozoa. Chlamydia trachomatis (CT) represents an intracellular microorganism responsible for sexually transmitted infections (STIs) in men and women. A reliable method of diagnosing this infection is therefore necessary because of the rapid onset of infection and the increase in STI-related diseases and their treatment costs. Methods: We analyzed 2371 semen samples from infertile men and detected the presence anti-CT IgG antibodies by Enzyme-Linked Immunosorbent Assay (ELISA), followed by real-time PCR confirmation of CT DNA whose target is the lipopolysaccharide (LPS). We assessed the effect of CT infections on characteristic parameters of sperm quality, including concentration, motility, viability, and morphology. The impact on sperm DNA quality was assessed by DNA fragmentation index (S) and decondensation of chromatin index (SDI) by the TUNEL technique. Results: Analysis of the results showed significant differences in mobility, concentration, and morphology (p < 0.05) between the control group, positive CT infection with normal spermiogram status (CT+/Normal SG) group, and positive CT infection with abnormal spermiogram status (CT+/Abnormal SG) group. A significant increase in the DFI and the SDI was found between the control group and the case groups, respectively (p < 0.01). Conclusions: Our results confirm that CT infection is associated with significant alterations in sperm parameters and sperm DNA quality. Regular CT screening by qPCR should be encouraged in couples suffering from unexplained infertility.