The burden of disease caused by Staphylococcus aureus continues to grow; this organism has the ability to form biofilm and it is also a frequent cause of medical device and implant-related infections. The objective of this study was to evaluate the biofilm-forming ability of a collection of clinical isolates of S. aureus. In a total of 240 Staphylococcus spp. isolated from catheters, retrieved at five services (neonatology, internal medicine, pneumology, pediatric and neurology), only 50 (20.83%) strains were identified by conventional microbiological methods as S. aureus species; these strains were screened by microtiter plate assay for detection of biofilm formation. Of the 50 clinical isolates, 16 (32%) were non adherent, 20(40%) weakly, 10 (20%) moderately and 4(8%) strongly adherent. The quantitative method of microtiter plate can be involved as a simple, rapid, inexpensive and reproducible assay to assess biofilm formation which is further an important feature of pathogenecity of S. aureus in the clinical setting.