The aim of this study was to evaluate the effects of tumour necrosis factor-alpha (TNF-alpha) on sperm motility, mitochondrial membrane potential (DeltaPsi), phosphatidylserine (PS) externalization, sperm chromatin packaging quality, and DNA fragmentation. Motile spermatozoa, obtained from 10 normozoospermic men, were incubated with increasing concentrations of TNF-alpha and analyzed 1, 3, 6, and 24 h after incubation by flow cytometry. TNF-alpha decreased total motility 24 h after incubation at 10 ng/mL and progressive motility 3 h after incubation. Accordingly, TNF-alpha reduced sperm DeltaPsi in a concentration- and time-dependent manner. TNF-alpha increased the percentage of spermatozoa with PS externalization from the concentration of 1 ng/mL 1 h after incubation. TNF-alpha produced sperm chromatin and DNA damage in a concentration- and time-dependent manner. In conclusion, these findings may explain the reduction of fertility, secondary to upregulated production of TNF-alpha, in men with urogenital infections.