The aim of the study was to evaluate the antioxidant potential of hydroxytyrosol (HT) on human sperm quality during incubation in vitro. Semen samples collected from men attending the Laboratory of Histology-Embryology of Sfax Faculty of Medicine (Tunisia) for infertility investigations were evaluated for initial sperm parameters. Only normal selected ejaculates (n = 15) were centrifuged and incubated further with or without HT (200ug ml ) at room temperature for 45 min. After incubation, sperm motility and viability, DNA oxidation and reactive oxygen species (ROS) production were assessed. The results showed that centrifugation significantly influenced sperm motility and viability. The supplementation of HT in incubating media improved (P = 0.01) significantly sperm viability and decreased sperm DNA oxidation (P < 0.001) and ROS levels (P = 0.03) following centrifugation. It can be concluded that supplementation of HT might be helpful to maintain the human spermatozoon after centrifugation.