Abstract Background The clinical landscape of prostate biopsy (PB) is evolving with changes in procedures and techniques. Moreover, antibiotic resistance is increasing and influences the efficacy of pre-biopsy prophylactic regimens. Therefore, increasing antibiotic resistance may impact on clinical care, which probably results in differences between hospitals. The objective of our study is to determine the (variability in) current practices of PB in the Netherlands and to gain insight into Dutch urologists’ perceptions of fluoroquinolone resistance and biopsy related infections. Methods An online questionnaire was prepared using SurveyMonkey® platform and distributed to all 420 members of the Dutch Association of Urology, who work in 81 Dutch hospitals. Information about PB techniques and periprocedural antimicrobial prophylaxis was collected. Urologists’ perceptions regarding pre-biopsy antibiotic prophylaxis in an era of antibiotic resistance was assessed. Descriptive statistical analysis was performed. Results One hundred sixty-one responses (38.3%) were analyzed representing 65 (80.3%) of all Dutch hospitals performing PB. Transrectal ultrasound guided prostate biopsy (TRUSPB) was performed in 64 (98.5%) hospitals. 43.1% of the hospitals (also) used other image-guided biopsy techniques. Twenty-three different empirical prophylactic regimens were reported among the hospitals. Ciprofloxacin was most commonly prescribed (84.4%). The duration ranged from one pre-biopsy dose (59.4%) to 5 days extended prophylaxis. 25.2% of the urologists experienced ciprofloxacin resistance as a current problem in the prevention of biopsy related infections and 73.6% as a future problem. Conclusions There is a wide variation in practice patterns among Dutch urologists. TRUSPB is the most commonly used biopsy technique, but other image-guided biopsy techniques are increasingly used. Antimicrobial prophylaxis is not standardized and prolonged prophylaxis is common. The wide variation in practice patterns and lack of standardization ...