Background: Cases with sepsis after transrectal ultrasound-guided prostate biopsy (TRUSPB) were documented, with special focus on cultures and susceptibility of isolates. We also evaluated the contribution of concomitant rectal cultures to the treatment of selective cases. Materials and Methods: Patients with sepsis after TRUSPB were followed prospectively. Manifestations and risk factors for antimicrobial resistance were documented. Results of urine and blood cultures and antimicrobial susceptibility were recorded for all participating patients. In 40 patients, rectal swab cultures were obtained concomitantly. Results: Ninety-five patients were consecutively studied. Sepsis symptoms started showing up within 48 h after biopsy in 93% of patients. At least one of the cultures was positive in 72 patients. E. coli grew in 70 cases; isolates were highly resistant: 67% displayed multidrug-resistance. Rectal cultures grew E. coli in 38 cases. In patients with positive E. coli in rectum and in at least one additional culture (blood and/or urine), the antibiogram was identical in all cases but one. Eight cases had negative cultures. Conclusion: The prevalence of antimicrobial resistance among E. coli isolates from patients with TRUSPB sepsis was alarming. Susceptibilities of rectal E. coli isolates used for deescalation of initial empiric treatment in culture-negative TRUSPB sepsis can contribute to the reduction of broad-spectrum antibiotics exposure.