The aim of this study was to evaluate the biocide and antimicrobial susceptibility of methicillin-resistant staphylococcal isolates from horses. Fourteen methicillin-resistant staphylococci (MRS) were subjected to an extensive genotype characterization, including SCCmec, dru, spa, PFGE and MLST typing. Antimicrobial susceptibility testing was performed and resistance genes were detected by PCR. Minimum bactericidal concentrations (MBCs) of four biocides [chlorhexidine acetate (CHA), benzalkonium chloride (BAC), triclosan (TCL) and glutaraldehyde (GLA)] were determined following the recommendations of document NF EN 1040. The presence of qac and sh-fabI genes was investigated by PCR. Several antimicrobial resistance patterns and genes were detected. When MRS strains were exposed for a longer period of time, a lower concentration of the biocide was needed to achieve lethality. TCL had the lowest MBC values. All MBC values were lower than the recommended in-use concentrations for veterinary medicine. S. haemolyticus and S. cohnii subsp. cohnii carried plasmid-borne qacA and sh-fabI or qacB and a qacH-like genes, respectively. Biocides appear to be a reliable antiseptic option against MRS, since even in the presence of bacterial efflux mechanisms, the recommended concentration is much higher than the in vitro MBC.