Background: Benign prostatic hyperplasia (BPH) is one of the most common reproductive disorders in both male dogs and men. Finasteride, a synthetic inhibitor of the enzyme 5α-reductase, is widely used as medical treatment. Although sperm can be affected by both BPH and finasteride treatment, the direct influence on DNA integrity remains unclear. Thus, the aim of this study was to verify the direct effect of BPH and/or finasteride treatment on DNA integrity of dog spermatozoa. A 2 × 2 factorial experiment was designed with 20 male dogs assigned to 4 experimental groups: BPH Group (n = 5), BPH-Finasteride Group (n = 5), Non-BPH Finasteride-Treated Group (n = 5) and Non-BPH Untreated Group (n = 5). Sperm evaluation was performed monthly for 60 days after the start of finasteride therapy or BPH diagnosis (D0, D30 and D60). Sperm DNA integrity was analyzed through fragmentation susceptibility (toluidine blue staining and Sperm Chromatic Structure Assay-SCSA), direct evaluation of DNA fragmentation (Sperm Chromatin Dispersion Assay-SCDA) and sperm protamination (chromomycin A3). Results: Sperm DNA integrity was not affected by finasteride treatment. However, BPH dogs had higher susceptibility to sperm DNA acid denaturation (SCSA) compared to dogs not presenting BPH, as well as lower percentage of sperm with DNA integrity (toluidine blue staining). Conclusion: In conclusion, benign prostatic hyperplasia causes post-testicular sperm DNA damage, albeit finasteride treatment itself does not directly influence sperm DNA integrity.