The clinical landscape of prostate biopsy 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 prostate biopsy in the Netherlands and to gain insight into Dutch urologists' perceptions of fluoroquinolone resistance and biopsy related infections. 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. One hundred sixty-one responses were analyzed representing 65 (80.3%) of all Dutch hospitals performing prostate biopsy.