Background: Transrectal ultrasound biopsy is the preferred method for diagnosing prostate cancer, but it can cause infectious complications as a result of fluoroquinolone resistance. We aimed to explore the potential protective effect of a second rectal enema before biopsy. Methods: Between January 2015 and December 2020, 419 patients were assessed retrospectively. Patients with a history of anticoagulant use, uncontrolled diabetes, urological surgery, prostate biopsy, or recent hospitalization or overseas travel, as well as those with previous prostatitis, were excluded from the study. The patients were subsequently divided into two groups: Group 1 (n=223) had received one enema, on the morning of the biopsy, and Group 2 (n=196) had received two, with the additional enema administered half an hour before the procedure. Results: There was no significant difference between the groups in terms of age, BMI, diabetes, prostate-specific antigen (PSA) level, and prostate size (p=0.076, p=0.489, p=0.265, p=0.193, and p=0.661, respectively) or in relation to cancer detection (p=0.428). The median hospitalization date was significantly higher in Group 1 (p=0.003) as was UTI development (p=0.004). However, there was no significant difference in terms of fever and sepsis (p=0.524 and p=0.548, respectively). Additionally, subgroup analysis demonstrated that UTI was significantly lower in patients with diabetes mellitus who had received a second enema (p=0.004), though there was no significant difference in UTI between the groups in those without diabetes mellitus (p=0.215). Multivariable analysis showed that age and diabetes were significant risk factors for the development of UTI (p=0.002andp=0.003, respectively). Furthermore, the second enema was a significant protective factor for preventing UTI (p<0.001). Conclusion: Older age and the presence of diabetes mellitus are independent risk factors for UTI after prostate biopsy. A second enema procedure before biopsy may protect patients from related infectious complications and could therefore be used as an alternative preventative method.