Staphylococcus epidermidis is the most prevalent coagulase-negative Staphylococcus (CNS) and is a major cause of hospital bacteremia. Based on 18 reference strains and 149 Staphylococcus clinical strains, used in a novel multiplex PCR method, the aim of this study was to identify S. epidermidis with respect to the sequence of three genes: recN, which encodes a recombination/repair protein, mecA (methicillin resistance), and icaAB, which is involved in biofilm formation. Amplicons of 219 bp (S. epidermidis-recN gene), 154 bp (mecA gene), and 546 bp (icaAB genes) were obtained. Reliable results were achieved for 100% of the evaluated strains, suggesting that this new multiplex-PCR approach could be useful for the accurate identification of methicillin-resistant S. epidermidis with the potential to produce biofilm.