<b><i>Background:</i></b> The purpose of this study is to compare the effect of different systems for eliminating duplicates in order to optimize the calculation of the prevalence of methicillin-resistant <i>Staphylococcus aureus</i> (MRSA) infection. <b><i>Methods:</i></b> We compare the Clinical and Laboratory Standards Institute (CLSI) criterion, time criteria and the criterion recommended by the European Antimicrobial Surveillance System (EARSS). <b><i>Results:</i></b> Multiple isolates of MRSA are frequently recovered from successive cultures from the same patient (the average isolation rate of MRSA is 2.72), which demonstrates the importance of eliminating duplicates. When CLSI criterion data are compared to those obtained using other criteria, a significant increase in the number of <i>S. aureus</i> isolates was found applying time criteria (up to 36%) or the EARSS criterion (13%). There is also an increase in the methicillin resistance rate (between 3.31 and 3.96%; p < 0.01). <b><i>Conclusions:</i></b> We believe that the EARSS method, with the proper quality controls and latest software tools available, is the best for determining the true situation of MRSA.