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The aim of the study was to assess the effect of timing of preoperative surgical antibiotic prophylaxis (SAP) on surgical site infection (SSI) and compare the different timing intervals.The benefit of routine use of SAP prior to surgery has long been recognized. However, the optimal timing has not been defined. For the purpose of developing recommendations for the World Health Organization guideline for SSI prevention, a systematic review and meta-analysis of all relevant evidence was conducted.Major medical databases were searched from 1990 to 2016. The primary outcome was SSI after preoperative-SAP comparing different timing intervals. Adjusted odds ratios (OR) with 95% confidence intervals (CI) were extracted and pooled for each comparison with a random effects model.Fourteen papers with 54,552 patients were included in this review. In a quantitative analysis, there was no significant difference when SAP was administered 120-60 minutes prior to incision compared to administration 60-0 minutes prior to incision. Studies investigating different timing intervals within the last 60 minutes time frame reported contradictive results. The risk of SSI almost doubled when SAP was administered after first incision (OR:1.89; 95%CI:[1.05-3.40]) and was 5 times higher when administered more than 120 minutes prior to incision (OR5.26; 95%CI:[3.29-8.39]).Administration of antibiotic prophylaxis more than 120 minutes before incision or after incision is associated a higher risk of surgical site infections than administration less than 120 minutes before incision. Within this 120-minute time frame prior to incision, no differential effects could be identified. The broadly accepted recommendation to administer prophylaxis within a 60-minute time frame prior to incision could not be substantiated.