Infectious complications following prostate biopsy are increasing and fluoroquinolone prophylaxis has recently been banned by the European Commission. In this systematic review we summarize the evidence for different antibiotic prophylaxis regimens. We searched MEDLINE®, Embase® and Cochrane Database for randomized controlled trials (inception to October 2019) assessing antimicrobial interventions in prostate biopsy. Primary outcome was infectious complications. Exclusion criteria were simultaneous interfering interventions. GRADE (Grading of Recommendations, Assessment, Development and Evaluations) was used to assess the certainty of evidence. Protocol was registered with PROSPERO (CRD42015026354). Overall 59 randomized controlled trials (14,153 participants) and 7 different antimicrobial interventions were included. Antibiotic prophylaxis reduced infectious complications compared to no prophylaxis (RR 0.56, 95% CI 0.40-0.77, p=0.0005, I2=15%, participants 1,753, studies 11). A short-term prophylaxis (single shot to 3 days) was inferior to a long-term prophylaxis (1 to 7 days) with fluoroquinolone (RR 1.89, 95% CI 1.37-2.61, p=0.0001, I2=0%, participants 3,999, studies 17). Fosfomycin trometamol was an alternative to fluoroquinolone with reduced rates of infectious complications (RR 0.49, 95 CI 0.27-0.87, p=0.02, I2=54%, participants 1,239, studies 3). Empiric prophylaxis was inferior to targeted prophylaxis (RR 1.81, 95% CI 1.28-2.55, p=0.0008, I2=48%, participants 1,511, studies 6). Standard prophylaxis was inferior to augmented prophylaxis (using multiple rather than single agent) using a fixed model (RR 2.10, 95% CI 1.53-2.88, p <0.0001, I2=71%, participants 2,597, studies 9), but not using a random model (p=0.07). No difference was observed in infectious complications based on route or timing of antimicrobial prophylaxis. The certainty of evidence was rated as low/very low. In countries where fluoroquinolones are allowed as antibiotic prophylaxis, a minimum of a full 1-day administration as well as targeted therapy in case of fluoroquinolone resistance is recommended. In countries with a ban on fluoroquinolones, fosfomycin is a good alternative, as is augmented prophylaxis, although no established standard combination exists to date.