Sperm DNA fragmentation index (DFI) assessed by sperm chromatin structure assay is a valuable tool for prediction of fertility in vivo. Previous studies on DFI as predictor of in vitro fertilization (IVF) outcome, based on relatively small materials, gave contradictory results. The present study examines, in a large cohort, the association between sperm DFI and the outcome of IVF/ICSI procedure. The study is based on 1633 IVF or ICSI cycles performed at the Reproductive Medicine Centre, Skåne University Hospital, Malmö, Sweden, between May 2007 and March 2013. DFI values were categorized into four intervals: DFI ≤ 10% (reference group), 10% < DFI ≤ 20%, 20% < DFI ≤ 30%, DFI > 30%. For the three latter intervals, the following outcomes of IVF/ICSI procedures were analyzed in relation to the reference group: fertilization, good quality embryo, pregnancy, miscarriage, and live births. In the standard IVF group, a significant negative association between DFI and fertilization rate was found. When calculated per ovum pick-up (OPU) Odds Ratios (ORs) for at least one good quality embryo (GQE) were significantly lower in the standard IVF group if DFI > 20%. OR for live birth calculated per OPU was significantly lower in standard IVF group if DFI > 20% (OR 0.61; 95% CI: 0.38–0.97; p = 0.04). No such associations were seen in the ICSI group. OR for live birth by ICSI compared to IVF were statistically significantly higher for DFI > 20% (OR 1.7; 95% CI: 1.0–2.9; p = 0.05). OR for miscarriage was significantly increased for DFI > 40% (OR 3.8; 95% CI: 1.2–12; p = 0.02). The results suggest that ICSI might be a preferred method of in vitro treatment in cases with high DFI. Efforts should be made to find options for pharmacologically induced reduction of DFI. The study was based on retrospectively collected data and prospective studies confirming the superiority of ICSI in cases with high DFI are warranted.