Methicillin-resistant Staphylococcus aureus (MRSA) emerged as one of the most important causes of hospital-acquired bloodstream infections (BSIs), especially the multidrug resistant clones. The aim of the present study was to compare prevalence and resistance patterns of MRSA bacteremia in the major tertiary-care academic and referral center in Serbia before and after implementing an active antimicrobial resistance (AMR) surveillance. Laboratory-based before-after study was conducted during a two-year period (January 2012 to December 2013) in Clinical Centre of Serbia. Isolation and identification of bacterial strains were done following standard microbiological procedures. During the AMR surveillance, nearly twice more bloodstream samples were collected compared to the year without surveillance (1,528 vs. 855). In total, 43 isolates of MRSA were identified. MRSA was significantly more prevalent during the AMR surveillance compared to the previous year [14 (66.7%) to 29 (76.3%); P = 0.046]. During the AMR surveillance, MRSA more frequently originated from medical departments compared to intensive care unit, surgical department, and internal medicine (P = 0.027) indicating increasing MRSA infections in patients with less severe clinical condition and no apparent risk factors. Higher prevalence of MRSA and its lower susceptibility to erythromycin were revealed by implementation of active AMR surveillance, which may reflect more thoughtful collection of bloodstream samples from patients with suspected BSI.