The in vitro killing effect of widely used antibiotics (cephalothin, clindamycin, erythromycin, ofloxacin, rifampicin, teicoplanin, tetracycline, phosphomycin and vancomycin) was comparatively analyzed in this study on 24-h biofilms of 64 Staphylococcus epidermidis clinical isolates. This effect was assessed at the expected antibiotic concentration reached in serum, using ATP-bioluminescence. Erythromycin, rifampicin, tetracycline and phosphomycin presented generally a higher killing effect than vancomycin, clindamycin, cephalothin, teicoplanin and ofloxacin in these biofilms. Differences in the resistance profiles obtained in classical assays (broth microdilution and diffusion) did not help to predict differences in the killing effect of the antibiotics in biofilms. Only some antibiotics (vancomycin but not rifampicin or tetracycline) highly decreased their killing effect as the biofilm age increased (from 6 to 24 or 48 h). These studies underline the relevance of biofilm susceptibility testing and the potential danger of the indiscriminate use of vancomycin monotherapy as the ultimate resource against infections involving aged biofilms.