Aims: To investigate bacterial contamination of environmental surfaces in An-Najah NationalUniversity setting. This study focused mainly on staphylococci bacteria in particular detection ofsome molecular characterization of Staphylococcus aureus.Place and Duration of the Study: Department of Biology and Biotechnology, An-Najah NationalUniversity, Palestine, March-August 2012.Methods: A total of 210 environmental surface samples from Faculty of Science, An-NajahNational University were collected using cotton swabs. S. aureus, biochemical tests and S. aureusspecies-specific identification was used to confirm the isolates. Antimicrobial susceptibility testingfor methicillin was performed, mecA gene, SCCmec typing and seg, seh and sei toxin genes weredetected by PCR. Morphology, biochemical tests and selective media were used to identify othermicroorganisms obtained from contaminated environmental surfaces.Results: It was found that 31.9% (67/210) of these surfaces were contaminated with S. aureus.Only 5 methicillin resistant S. aureus (MRSA) isolates were detected and belonged to SCCmec IVatype. One isolate of methicillin sensitive S. aureus (MSSA) was enterotoxigenic and had genotype seg+/seh+. In addition, 85.7%, 90.5%, 14.3%, 11.9%, 10% and 4.8% of cultures were positive for S. epidermidis, fungi, Bacillus sp., Escherichia coli, Klebsiella sp. and Streptococcus sp., respectively. Conclusion: The results show that these different representative environmental surfaces are in daily use and may be a source of infection.