Objective: Transrectal ultrasound-guided prostate (TRUS) biopsy is the standard method for the diagnosis of prostate cancer. Quinolone-based prophylaxis before a TRUS biopsy of the prostate is the most common regimen worldwide. In this retrospective study, we evaluated the efficacy and cost effectiveness of adding gentamicin to a fluoroquinolone-based prophylaxis regimen. Materials and methods: In total, our study included 263 patients classified into two groups. Group 1 consisted of 129 patients who received one oral dose of levofloxacin (500 mg) daily 2 days before the biopsy, on the day of the biopsy, and for 2 days after the biopsy. Group 2 consisted of 134 patients who received a single intramuscular (IM) gentamicin injection (80 mg) 30 minutes before the biopsy in addition to the same oral levofloxacin protocol as Group 1. We recorded and analyzed data including age, indication for a TRUS biopsy of the prostate, prostate volume, comorbidity, infectious complications, and blood and urine culture results. Results: The mean prostate-specific antigen level was 38.6 ng/mL ± 312.9 ng/mL (range, 4.4–2626 ng/mL) in Group 1, and 34.8 ng/mL ± 127.1 ng/mL (range, 2.11–1423 ng/mL) in Group 2. The groups were similar in terms of mean age, indication for a biopsy, prostate volume, number of biopsy cores taken, and comorbidities. Infection-related complications occurred in eight of 129 (6.2%) and in one of 134 (0.74%) patients in Groups 1 and 2, respectively (p = 0.02). Conclusion: The addition of IM gentamicin (80 mg) was beneficial in improving the efficacy of fluoroquinolone and reducing the post-TRUS biopsy infection rate. Gentamicin is relatively inexpensive and readily available in daily practice and has good compliance for patient use.