Staphylococcus aureus is one of the most important etiological agents responsible for healthcare-associated infections and is capable of producing many virulence factors including biofilm. The aim of the present study was to analyze the correlation between the presence of the icaD and icaA genes and the ability to produce biofilm in vitro in 302 methicillin-resistant (MRSA) and 268 methicillin-sensitive S. aureus (MSSA) strains isolated in the Provincial Hospital in Gdansk. Presence of the icaD and icaA genes was detected by PCR and the ability to produce biofilm in vitro was measured both spectrophotometrically and via Congo Red Agar plate culture methods. We found that 91% of MRSA strains harbored the icaD gene. Moreover, all icaD-negative strains were icaA-positive. Of MRSA and MSSA strains, 47% and 69%, respectively, produced biofilm in vitro. The level of consistency between the two applied phenotypic methods was 96%. Additionally, we found that strains with the same biofilm status may be present in asymptomatic carriers and cause infections.