Purpose <p>To evaluate the impact of omitting preoperative biopsy on postoperative functional outcomes and health-related quality of life (HRQOL) in patients undergoing radical prostatectomy (RP).</p> Methods <p>This retrospective cohort study used data from the prostatectomy database at LMU University Hospital. Patients were divided into a biopsy group (<i>n</i> = 8995) and a non-biopsy group (<i>n</i> = 68). Propensity score matching was performed to control for confounders, resulting in a matched cohort (biopsy group <i>n</i> = 186, non-biopsy group <i>n</i> = 68). Urinary continence (ICIQ-SF, pads/day), sexual function (IIEF-5), and HRQOL (EORTC QLQ-C30) were compared between the two groups at multiple time points (preoperatively and at 3–48 months postoperatively) in matched and unmatched cohorts, and also in pT2 subgroups.</p> Results <p>All patients in the non-biopsy group had highly suspected prostate cancer and were pathologically confirmed to have significant prostate cancer. In the matched cohort, HRQOL was significantly better in the biopsy group at 3 months. In the matched cohort, the non-biopsy group showed no advantage regarding continence and sexual function, but higher symptom burden at several time points. Similar trends were observed in the pT2 subgroups.</p> Conclusion <p>This study showed no evidence of a clinically relevant difference regarding functional or HRQOL outcome after RP between patients with and without preoperative prostate biopsy.</p>