Objectives: Transrectal ultrasound guided prostate biopsy (TRUSPB) is the standard of care for diagnosis of prostate cancer. Increased antibiotic resistance has led to the suspension of indication for fluoroquinolones use in prostate biopsy prophylaxis. Several classes of antibiotics have been recommended for routine use. Unequivocal consensus regarding antibiotic prophylaxis has not been made to date. The objective of the study was to assess the diversity of antimicrobial prophylaxis among Croatian urologists. Materials and methods: An online questionnaire was designed using Google Forms® and distributed to 19 urology public hospital’s departments. Answers regarding infection risk assessment, type and duration of antimicrobial prophylaxis were accumulated. Descriptive statistical analysis was preformed using Statistica 10.0® analytics software. Results: Twelve urology departments answered the questionnaire, representing 63% of urology departments in Croatia. Six different antibiotic protocols have been reported. Fluoroquinolones were the most commonly prescribed class of antibiotics (84%). Antibiotic prophylaxis started 1 day before the procedure (92%). Average duration of antibiotic prophylaxis was 5 days (75%). In case of increased risk of urinary tract infection, 42% of departments changed the type, and 8% changed the duration of antibiotic prophylaxis. Neither department performed a rectal swab prior to prostate biopsy. Conclusions: Various antimicrobial prophylaxis protocols are currently being used among Croatian urology departments. Lack of uniform guidelines contributes to protocol diverseness that inevitably leads to further increase in antibiotic resistance. New high quality studies are needed to reverse this trend and to facilitate the establishment of a uniform antimicrobial stewardship strategy.