Intrauterine insemination with donor sperm (IUI-D) requires multiple in vitro manipulations such as sperm selection and cryopreservation during which spermatozoa may be exposed to oxidative stress (OS) and other insults that may produce potential damage including sperm DNA fragmentation (SDF). High levels of SDF, referring to damage or breaks in the genetic material of sperm cells, are linked to an increased risk of reproductive failure. This retrospective, observational study set out to evaluate whether SDF assessment could predict clinical outcome in an IUI-D program, where sperm donors are selected on strict conventional semen parameters. A total of 18 donors and 106 recipients were matched for IUI-D. Out of 429 cycles, 100 (23.3%) resulted in clinical pregnancy. We counted 78 live births (18.2% of cycles), while 20 pregnancies ended in miscarriage (4.7% of cycles), 1 in extra-uterine pregnancy and 1 in stillbirth. Female age significantly influenced clinical pregnancy and miscarriage rates. SDF increased after cryopreservation (26.3 ± 14.5%; p < 0.001) and more so after post-thaw density gradient (34.9 ± 22.1%; p = 0.04) without affecting clinical pregnancy (OR [95% CI] 1.01 [0.99; 1.02]; p = 0.27), live birth (1.00 [0.99; 1.02]; p = 0.72) and miscarriage rates (1.02 [1.00; 1.05]; p = 0.08). The implications of our findings extend to a better selection of sperm donors and a better sperm preparation technique tailored to the donor semen’s properties in order to maximize the chances of a favorable treatment outcome.