The incidence of Ureaplasma urealyticum infection in the semen of infertile men is variable (7%–42%). Evidence has accumulated through routine semen analysis to suggest that this infection can cause embryo loss without necessarily affecting sperm quality. The aim of this study was to specifically investigate the effects of U. urealyticum infection on sperm chromatin stability and DNA integrity, which are known to be correlated to pregnancy outcome. Sperm cells isolated from human semen infected in vivo with U. urealyticum exhibited a low percentage of stable chromatin as determined by nuclear chromatin decondensation assay (42% ± 4.8%, n = 8) and a high percent of denatured DNA as determined by sperm chromatin structure assay (60.9% ± 9.1%, n = 7). After doxycyclin treatment, a significant improvement in both parameters was observed (73.7% ± 3.6%, P < 0.001 and 30.1% ± 3.5%, P < 0.008, respectively). Sperm cells infected in vitro exhibited higher rates of viability and motility than uninfected cells. In contradistinction, U. urealyticum caused significant dose- and time-dependent chromatin decondensation and DNA damage. The percentage of human sperm cells with denatured DNA increased significantly by 54.9% ± 23.9% and 47.9% ± 12.1%, after 30 min infection with serotypes 8 and 3, respectively, at a multiplicity of infection of 100 ureaplasmas per sperm compared with uninfected control cells. The damage to DNA was significantly more pronounced in infected ram sperm (180.9% ± 21.5%). These results indicate that preserved sperm activity post U. urealyticum infection resulted in damage to paternal DNA, although a high fertilization rate was maintained, and embryonic development may, therefore, be impaired.