Background: Infectious complications after a transrectal prostate biopsy may be severe. In Sweden, a routine culture prior to all prostate biopsies was introduced to enable targeted antimicrobial prophylaxis and reduce postbiopsy infections. Objective: To investigate whether a clinical routine with a urine culture prior to a prostate biopsy and targeted prophylactic antibiotic therapy reduces postbiopsy infections. Design, setting, and participants: In 2015, a site-specific antimicrobial stewardship programme with a urine culture prior to a prostate biopsy was initiated in Region Kronoberg. To evaluate this routine, we designed a population-based register study including all men who had an outpatient prostate biopsy in 2015–2019 and a control period including all men who had a biopsy in 2010–2014, when a urinary culture was obtained only on clinical suspicion. Outcome measurements and statistical analysis: The primary outcome was infectious complications within 10 d and the secondary outcome was a change in antibiotic prophylactic treatment. An infectious complication was defined as prescription of antibiotics for urinary tract infections or admission to hospital for urinary tract infections or sepsis after a biopsy. Results and limitations: The urine culture period included 2971 prostate biopsy procedures, of which 2684 (90%) were preceded by a urine culture. The control period included 2818 procedures, of which 135 (4.8%) were preceded by a urine culture. Infectious complications were slightly more common during the urine culture period (5.0%) than during the control period (4.3%, p = 0.17), as was inpatient care for infections (3.5% vs 2.2%, p = 0.002). The routine identified 5.4% men with asymptomatic bacteriuria. Despite targeted antibiotic treatment (1.5% received a nonfluoroquinolone treatment), the rate of infectious complications (6.3%) was similar to that in the control period. Conclusions: Prebiopsy urine culture did not lead to fewer postbiopsy infections. Other measures are needed to reduce infectious complications after a prostate biopsy. Patient summary: In this report, we evaluated a routine with urine culture prior to a transrectal prostate biopsy and found that it did not lead to fewer infectious complications.