Biofilm resistance mechanisms are multifactorial and vary from one organism to another. The purpose of this study was to investigate linezolid's efficacy against indwelling device-related methicillin-resistant Staphylococcus epidermidis (MRSE) biofilm, and compared with others antimicrobials. Minimal inhibitory concentrations (MICs), minimum biofilm inhibitory concentrations (MBICs) and minimum biofilm eradication concentrations (MBECs) were determined by the microtiter plate method. Fourteen and thirteen isolates from patients with indwelling device-related bacteremia and indwelling device colonization, respectively, were assessed. High MBIC was associated with high intensity of biofilm formation (gentamicin r = 0.796; linezolid r = 0.477; rifampicin r = 0.634; tigecycline r = 0.410 and vancomycin r = 0.771), but this correlation was not observed with MBEC. Linezolid demonstrated better in vitro antimicrobial activity than other antimicrobials (MBIC: gentamicin P < 0.001, rifampicin P = 0.019, vancomycin P = 0.008; MBEC: gentamicin P < 0.001, rifampicin P = 0.002, vancomycin P < 0.001). Biofilm growth inhibition was strongly associated with biofilm formation intensity; however biofilm eradication was not cell number dependent. MRSE biofilms eradication would represent a huge advance for indwelling device-related bacteremia, although high concentrations of gentamicin, linezolid, rifampicin, tigecycline and vancomycin were required for that. In general, linezolid reached better in vitro concentrations and demonstrated to be highly active against MRSE biofilms by inhibiting their growing when forming biofilm.