Background: Biofilm formation capacity is recognized as an important virulence factor in staphylococci that makes the organisms more resistant to antibiotics and host defenses. Objectives: This study aimed to determine the biofilm producing ability and presence of icaA/D genes in staphylococcal isolates obtained from different clinical specimens. Methods: This cross-sectional study was performed on a total of 151 staphylococcal isolates (79 Staphylococcus aureus and 72 S. epidermidis) obtained from different clinical specimens from February to August 2013 in Shiraz, Southwest of Iran. Slime production ability was evaluated using the both phenotypic (by cultivation of staphylococcal isolates on Congo red agar (CRA)) and genotypic (detection of the presence of icaA/D genes by PCR) methods. Results: Overall, of the 79 S. aureus isolates tested with CRA method, 64.7% of methicillin-resistant S. aureus (MRSA) isolates, and 46.7% of methicillin-sensitive S. aureus (MSSA) isolates were able to produce biofilm. The relative frequency of biofilm producing S. epidermidis isolates was 70.8% that was significantly higher than that of S. aureus isolates. The most common source of biofilm producing isolates in both S. aureus and S. epidermidis isolates was endotracheal tube (ETT) with 100% biofilm formation. Moreover, the presence of icaA/D genes was detected in 63.3% and 81.9% of S. aureus and S. epidermidis isolates, respectively. Conclusions: The remarkable rates of biofilm production ability among clinically isolated staphylococci emphasize the necessity of more effective infections control policies to prevent biofilm formation on medical devices and hospital environmental surfaces.