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Biofilms are clusters of microorganisms that form at various interfaces, including those between air and liquid or liquid and solid. Due to their roles in enhancing wastewater treatment processes, and their unfortunate propensity to cause persistent human infections through lowering antibiotic susceptibility, understanding and managing bacterial biofilms is of paramount importance. A pivotal stage in biofilm development is the initial bacterial attachment to these interfaces. However, the determinants of bacterial cell choice in colonizing an interface first and heterogeneity in bacterial adhesion remain elusive. Our research has unveiled variations in the buoyant density of free-swimming Staphylococcus aureus cells, irrespective of their growth phase. Cells with a low cell buoyant density, characterized by fewer cell contents, exhibited lower susceptibility to antibiotic treatments (100 μg/mL vancomycin) and favored biofilm formation at air–liquid interfaces. In contrast, cells with higher cell buoyant density, which have richer cell contents, were more vulnerable to antibiotics and predominantly formed biofilms on liquid–solid interfaces when contained upright. Cells with low cell buoyant density were not able to revert to a more antibiotic sensitive and high cell buoyant density phenotype. In essence, S. aureus cells with higher cell buoyant density may be more inclined to adhere to upright substrates.