Coagulase-negative Staphylococcus spp. (CNS) has been associated with primary bloodstream infections and implanted medical devices. Its importance is increasing due to the acquisition of resistance to oxacillin (Oxa) and, recently, resistance to mupirocin (Mup). Mupirocin, a topical antimicrobial, has been used in the prevention of staphylococci catheter colonization. Susceptibility to Oxa and Mup was analyzed by different testing methods in clinical CNS isolates. Among 112 CNS strains, 69 (61.6%) were Oxa(R) by the disk diffusion (DD) method and 72 (64.2%) grew on the oxacillin agar screen plate. S. epidermidis and S. haemolyticus presented high rates of oxacillin resistance, 75.4% and 96.1%, respectively. Twenty four (21.4%) strains were Mup(R) by the DD test and 21 of them (87.5%) were identified as S. epidermidis. The detection of the mecA and ileS-2 genes, determined by multiplex-PCR, showed that 72 (64.2%) CNS strains possessed the mecA gene, while 16 (14.3%) possessed the ileS-2 gene. Fifteen of these strains presented the two resistance genes simultaneously. The isolates containing the ileS-2 gene presented a minimum inhibitory concentration (MIC) >1024 microg/mL in the E-test, while low-level mupirocin resistance (MICs of 12-16 microg/mL) was observed in those strains without ileS-2. The resistances to high and low levels of mupirocin could not be distinguished when the DD test was used. The analysis of the Mup(R) S. epidermidis strains by Pulsed Field Gel Electrophoresis showed that 17 (80.9%) strains belonged to one of two patterns (A and B), which have been shown to be prevalent in hospitals in Rio de Janeiro. This report showed that the PCR method for detection of oxacillin and mupirocin resistance in CNS is necessary to determine accurate rates of these resistance, and will can help in the staphylococcal infections prevention and control policies in Brazil.