Objective: The objective of this study was to determine the prevalence of antibiotic resistance in fecal flora of patients undergoing transrectal ultrasound-guided needle biopsy of the prostate (TRUSB) and the factors associated with such antibiotic resistance. Methods: A prospective study of patients undergoing TRUSB was conducted. Rectal swabs were performed and sent for cultures and antibiotic susceptibility testing before TRUSB. Clinical characteristics were determined. Results: 287 Gram-negative isolates from 144 patients were identified, 80.1% were Escherichia coli and 13.9% were Klebsiella pneumoniae. 27 patients who received antibiotics within 3 months exhibited higher prevalence of organisms with extended-spectrum beta-lactamases (ESBL) production (40.7 vs. 22.2%) and ceftriaxone-resistance (48.1 vs. 28.2%). 134 patients received a short-course antibiotic prophylaxis in which fluoroquinolone (FQ) contributed to 89.6% of cases. Patients who received antibiotic prophylaxis showed a higher prevalence of organisms resistant to ceftriaxone (34.3 vs. 0%), ciprofloxacin (90.3 vs. 30%) and FQ (95.5 vs. 50%) and a trend of more ESBL production (27.6 vs. 0%). Conclusions: Previous antimicrobial use and prophylaxis with FQ are correlated with a higher prevalence of FQ and ceftriaxone resistance and ESBL production. A single dose of ceftriaxone without short-course FQ use is recommended as antibiotic prophylaxis in TRUSB.