OBJECTIVE: To evaluate the effectiveness of an intervention based on training and the use of a protocol with an automatic stop of antimicrobial prophylaxis to improve hospital compliance with surgical antibiotic prophylaxis guidelines. DESIGN: An interventional study with a before-after trial was conducted in 3 stages: a 3-year initial stage (January 1999 to December 2001), during which a descriptive-prospective survey was performed to evaluate surgical antimicrobial prophylaxis and surgical site infections; a 6-month second stage (January to June 2002), during which an educational intervention was performed regarding the routine use of a surgical antimicrobial prophylaxis request form that included an automatic stop of prophylaxis (the "automatic-stop prophylaxis form"); and a 3-year final stage (July 2002 to June 2005), during which a descriptive-prospective survey of surgical antimicrobial prophylaxis and surgical site infections was again performed. SETTING: An 88-bed teaching hospital in Entre Ríos, Argentina. PATIENTS: A total of 3,496 patients who underwent surgery were included in the first stage of the study and 3,982 were included in the final stage. RESULTS: Comparison of the first stage of the study with the final stage revealed that antimicrobial prophylaxis was given at the appropriate time to 55% and 88% of patients, respectively (relative risk [RR], 0.27 [95% confidence interval {CI}, 0.25-0.30]; P<.01); the antimicrobial regimen was adequate in 74% and 87% of patients, respectively (RR, 0.50 [95% CI, 0.45-0.55]; P<.01); duration of the prophylaxis was adequate in 44% and 55% of patients, respectively (RR, 0.80 [95% CI, 0.77-0.84]; P<.01); and the surgical site infection rates were 3.2% and 1.9%, respectively (RR, 0.59 [95% CI, 0.44-0.79]; P<.01). Antimicrobial expenditure was 10,678.66 US$ per 1,000 patient-days during the first stage and 7,686.05 US$ per 1,000 patient-days during the final stage (RR, 0.87 [95% CI, 0.86-0.89]; P<.01). CONCLUSION: The intervention based on training and application of a protocol with an automatic stop of prophylaxis favored compliance with the hospital's current surgical antibiotic prophylaxis guidelines before the intervention, achieving significant reductions of surgical site infection rates and substantial savings for the healthcare system.