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Fluoroquinolones (FQs) have been traditionally used for prophylaxis against bacterial infection. However, the rapid emergence of FQ-resistant Escherichia coli due to overuse and misuse have resulted in an increase in post-biopsy infections. We requested 723 patients undergoing transrectal or transrectal plus transperineal targeted prostate biopsy to provide preprocedure rectal swabs. The rectal swabs were plated onto deoxycholate hydrogen sulfate lactose agar culture and FQ resistance tests were conducted using the disc diffusion method following the guidelines of the Clinical and Laboratory Standards Institute. All patients undergoing biopsy were given a 1.0 g intravenous injection of cefmetazole (CMZ) 30 min before and 12 h after biopsy. Patients with FQ-resistant organisms received an additional 1.0 g intravenous injection of CMZ every 12 h for an additional 1.5 days, while those without FQ-resistant organisms received levofloxacin 500 mg for 4 days. We evaluated infectious symptoms during the 30 days after the biopsy. We also evaluated the incidence of acute prostatitis within 7 days after the biopsy and isolation rates of FQ-resistant strains. A total of 289 patients (40%) had FQ-resistant isolates on rectal swabs. The overall infectious complication rate was 0.69%. Two patients with FQ-resistant isolates and three patients without them experienced infectious episodes. One patient with FQ-resistant isolates and two patients without them suffered acute prostatitis. The difference in the rates of infectious complication and acute prostatitis rates between FQ-resistant and FQ-susceptible carriers were not significant (p = 1.0 and 1.0, respectively). Post-biopsy sepsis was identified in one patient (0.14%) who had FQ-resistant Escherichia coli. Targeted antimicrobial prophylaxis with cefmetazole based on presence of FQ-resistant isolates on rectal swabs may prevent post-prostate biopsy infectious complications, especially in geographic lesions with a high incidence of FQ-resistant strains in rectal flora.