Fifteen percent of male infertility is associated with urogenital infections; several pathogens are able to alter the testicular and accessory glands’ microenvironment, resulting in the impairment of biofunctional sperm parameters. The purpose of this study was to assess the influence of urogenital infections on the quality of 53 human semen samples through standard analysis, microbiological evaluation, and molecular characterization of sperm DNA damage. The results showed a significant correlation between infected status and semen volume, sperm concentration, and motility. Moreover, a high risk of fragmented sperm DNA was demonstrated in the altered semen samples. Urogenital infections are often asymptomatic and thus an in-depth evaluation of the seminal sample can allow for both the diagnosis and therapy of infections while providing more indicators for male infertility management.