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To characterize the pattern of surgical antimicrobial prophylaxis in the Czech Republic.Cross sectional survey with a self-administered postal questionnaire. Data collected included use of antimicrobial prophylaxis, surgical site infection rate, pathogens causing surgical site infection and demographics of the institution. Descriptive and multivariate analyses were performed.Hospital, surgical departments in the Czech Republic.Prevalence of surgical antimicrobial use, factors associated with use, the profile of antimicrobial use, timing, route, dosage regimen and duration of initiated prophylaxis.The response rate was 55.5%. Surgical antimicrobial prophylaxis was used in 97.5% of departments, and 85% departments justified prophylaxis based on guideline. The timing of the first dosage was within 2 h of operation in 95.0% of departments and 36.7% of all departments administered more than 2 doses of SAP in operations that lasted less than 4 h of all respondents. The three most common prophylactic antimicrobial agent used were cefazolin, co-amoxiclav and cefuroxime amongst the 26 single antimicrobial agents and 16 antimicrobial combinations. Penicillins and enzyme inhibitor was the most frequent class used. Surgical antimicrobial prophylaxis was administered intravenously in 82.5% of all cases. The regimen used varied markedly in dose and duration prescribed. The surgical site infection rate occurred. 1–5% in 71.7% of departments. Most departments identified the causative pathogen at all times. Staphylococcus aureus was the most frequent pathogen of surgical site infection and was detected in 90.8% of all departments. There was significant association between Pseudomonas aeruginosa with cefuroxime use and Bacteriodes fragilis with co amoxiclav use.This survey has identified several areas for improvement in surgical antimicrobial prophylaxis in the Czech Republic. Particular areas of concern include route of administration, duration and timing of first dosage of SAP, and the inappropriate use of broad-spectrum antimicrobials.