A total of 128 isolates associated with catheter-related infections was recovered from 101 intensive care unit patients in a Spanish hospital during March 2008 to August 2009, and 27 of these isolates (from 21 patients) were typed as methicillin- and linezolid-resistant Staphylococcus epidermidis. Thirteen of these 21 patients (62%) had received linezolid during the 3 months preceding S. epidermidis recovery. Two closely related pulsotypes (P1a and P1b) were identified among the 27 studied isolates that belonged to the sequence type ST2 and harboured the mecA gene and the SCCmecIII type. The strains recovered from patients 1-4 (pulsotype P1a) showed the nucleotide mutation G2474T inside the amplified fragment of the 23S rRNA region and carried the aac(6')-Ie-aph(2″)-Ia, ant(4'), and catA genes, whereas the strains from patients 5-21 (pulsotype P1b) showed the mutation G2603T and carried the aac(6')-Ie-aph(2″)-Ia gene. None of the strains amplified the cfr gene. The ica gene and the IS256 element were detected in all strains. The emergence of 2 closely related methicillin- and linezolid-resistant S. epidermidis strains with 2 different mutations in the 23S rRNA region (G2474T and G2603T) is reported in this study as a cause of a nosocomial outbreak. The presence of G2474T or G2603T point mutations suggests that there are multiple potential sites within domain V of the 23S rRNA region at which mutations could confer resistance to linezolid in the clinical isolates. To our knowledge, this is the first report in which the G2474T mutation has been detected in domain V of the 23S rRNA gene of clinical S. epidermidis.