The goal of this study was to investigate the effect of growth conditions such as the temperature (20, 30 and 37 °C), incubation duration (24 and 48 h) and surface type (stainless steel and polycarbonate) on the cell surface physicochemical properties and adhesion to abiotic surfaces of biofilm-detached and planktonic Staphylococcus aureus cells. This study tested also the hypothesis that S. aureus planktonic cells exhibit distinct pathogenic properties compared with their sessile counterparts. The results showed that the changes of the growth conditions promoted changes in the zeta potential, hydrophobicity, electron donor/acceptor character of the studied cell populations. Biofilm-detached cells showed a greater adhesion to stainless steel and polycarbonate compared with planktonic cells. Compared with planktonic cells, sessile ones showed higher cytotoxic effect against HeLa cells, DNase activity, and siderophore levels. The higher cytotoxic effect and production of DNase and siderophore increased with the increase of temperature and duration of incubations. Based on the obtained data, the S. aureusbiofilm-detached cells were found to be distinct in many physiological properties compared with their planktonic counterparts.