The literature suggests an association between sperm DNA damage and assisted reproductive technology (ART) outcomes. However, previous studies involved the transfer of multiple embryos, which has complicated the interpretation of the results. The aim of this study was to determine the relationship between the levels of sperm DNA damage and fertilization rate, embryo development as well as pregnancy outcome, following single embryo transfer. Patients (n = 113) undergoing in vitro fertilization (IVF) (n = 45) and intra-cytoplasmic sperm injection (ICSI) (n = 68) were assessed for their levels of sperm DNA damage in the sample used for insemination. DNA damage was determined using terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-nick end labelling (TUNEL). The relationship between DNA damage and outcomes were assessed using regression analysis. Overall data showed no association between sperm DNA damage and fertilization rate, or embryo development in vitro. However, when IVF was the insemination method, there was a significant negative correlation between fertilization rates and sperm DNA damage (p < 0.05). When ICSI was the insemination technique, low sperm DNA damage was associated with successful pregnancy (37.8 +/- 5.7% DNA damaged sperm) compared with failed implantation (52.9 +/- 3.9% DNA damaged sperm, p < 0.05). Our results suggest that sperm DNA damage as measured by the TUNEL assay may provide an indicator for patients with poor fertilization rates and/or those unable to achieve pregnancy following ART treatment.