Background: Biofilm formation plays an important role in resistance of Staphylococcus aureus isolates; especially multidrug-resistant isolates are a threat to healthcare settings. Objectives: The aims of this study were to detect biofilm formation and presence of several related genes among multidrug-resistant (MDR) isolates of Staphylococcus aureus. Patients and Methods: A total Of 209 S. aureus strains were isolated from patients and identified by conventional diagnostic tests. The multidrug-resistant MRSA isolates were detected by antibiotic susceptibility test. The phenotypic biofilm formation was detected by microtitre tissue plate assay. The polymerase chain reaction (PCR) was performed to detect the mecA, Staphylococcal Cassette Chromosome mec (SCCmec) types, accessory gene regulatory (agr) genes, the icaADBC and several genes encoding staphylococcal surface proteins including clfAB, fnbAB, fib, eno, can, ebps and bbp genes with specific primers. Results: Sixty-four (30.6%) isolates were methicillin-resistant, among which thirty-six (56.2%) were MDR. These isolates were resistant to amoxicillin, tetracycline, ciprofloxacin, gentamicin, erythromycin and trimethoprim-sulfamethoxazole (except to 6 isolates). All the isolates were susceptible to vancomycin and linezolid. All the MDR-MRSA harbored SCCmec type III. All the MDR-MRSA isolates were strong biofilm producers in the phenotypic test. The majority of MDR-MRSA was belonged to agrI (67%, n = 24), followed by agr II (17%, n = 6), agrIV (11%, n = 4) and agrIII (5.5%, n = 2). The frequency of icaADBC genes were 75% (n = 27), 61% (n = 22), 72% (n = 26) and 72% (n = 26), respectively. Furthermore, the prevalence of clfA, clfB, fnbA, fnbB, fib, can, eno, ebps and bbp genes was 100%, 100%, 67%, 56%, 80%, 63%, 78%, 7% and 0%, respectively. Furthermore, approximately all the MRSA was strong biofilm producers. Conclusions: Multidrug-resistant isolates produced biofilm strongly and the majority harbored most of biofilm related genes, suggesting that biofilm formation is associated to the presence of these genes, and also biofilm production can increase the antibiotic resistance as have demonstrated in MDR-MRSA isolates.