Background Prostate cancer is the most common malignant solid tumour in men aged >70 years and is the second most common cause of death from oncological circumstances. Aim To evaluate the effect of different short-term prophylactic antibiotic regimens in transrectal prostate biopsy (PB) on the incidence of infectious complications. Methods Patients who underwent transrectal ultrasound-guided PB between January 2021 and December 2022 were included in the prospective randomized study. According to the regimen of prophylaxis, patients were randomized into three groups: (1) fosfomycin trometamol 3 g, 3 h before the procedure + ciprofloxacin 500 mg, 2 h before the procedure; (2) fosfomycin trometamol 3 g, 3 h before and 24 h after the procedure; (3) ciprofloxacin 500 mg 12, 2 h before the procedure, and 12 h after the procedure. A rectal swab was performed 1–2 weeks before PB to evaluate the culture findings. Complications were evaluated during follow-up visits within one month after PB. Findings In the monitored period, 605 PBs were performed, and 544 patients met the inclusion criteria (184, 161, and 199 in groups 1, 2, and 3). Infectious complications occurred in 10 cases (1.83%), namely 3, 4, and 3 according to patient groups. There was no statistically significant difference between the individual groups. None of the patients required hospitalization and all were free of symptoms of sepsis. Conclusion Short-term antibiotic prophylaxis in PB using fosfomycin trometamol, ciprofloxacin, or their combination appears to be effective. Fosfomycin trometamol is a suitable alternative to fluoroquinolone antibiotics.