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Biofilm-mediated infections in the hospital environment have a significant negative impact on patient health. This study aimed to investigate biofilm production in vitro and the presence of icaABCD genes in methicillin-resistant S. aureus (MRSA) and methicillin-sensitive S. aureus (MSSA) strains isolated from hospitalized patients. MRSA (73) and MSSA (57) strains were evaluated for biofilm production by the microtiter plate method. The presence of ica operon was investigated by PCR. Out of 130 strains, 99.2% were biofilm producers. Strong biofilms were formed by 39.7% of MRSA and 36.8% of MSSA strains. The highest percentage of strong biofilm producers was found among the strains isolated from sputum and tracheostomy tube (66.7%), nose and catheter (50%), throat (44.4%), and bronchoalveolar washings (43.8%). The strains isolated from bronchoalveolar washings produced significantly more biofilm than strains isolated from wound and anus. The ability of biofilm forming by fecal strains was significantly lower compared to strains from other materials. MRSA strains had significantly higher ability of biofilm formation than MSSA strains (P = 0.000247). The presence of ica operon in MRSA was detected in all strains. Comparison of strong biofilm biomass of the strains with icaABCD, icaABD, and icaAD revealed that strains with icaABCD and icaABD produced highly significantly more biofilm than strains with icaAD. Biofilm forming by both MRSA and MSSA strains indicates high ability of theses strains to persist in hospital environment which increases the risk of disease development in hospitalized patients. [ABSTRACT FROM AUTHOR]