Formation of non-sessile, auto-aggregated cells of Staphylococcus aureus contributes to surface colonization and biofilm formation, hence play a major role in the early establishment of infection and in tolerance to antimicrobials. Understanding the mechanism of aggregation and the impact of aggregation on the activity of antimicrobials is crucial in achieving a better control of this important pathogen. Previously linked to biological phenomena, physical interactions leading to S. aureus cellular aggregation and its protective features against antimicrobials remain unraveled. Herein, in-vitro experiments coupled with XDLVO simulations reveal that suspensions of S. aureus cells exhibit rapid, reversible aggregation (> 70%) in part controlled by the interplay between cellular hydrophobicity, surface potential and extracellular proteins. Changing pH and salt concentration in the extracellular media modulated the cellular surface potential but not the hydrophobicity which remained consistent despite these variations. A decrease in net cellular negative surface potential achieved by decreasing pH or increasing salt concentrations, caused attractive forces such as the hydrophobic and cell–protein interactions to prevail, favoring immediate aggregation. The aggregation significantly increased the tolerance of S. aureus cells to quaternary ammonium compounds (QAC). The well-dispersed cell population was completely inactivated within 30 s whereas its aggregated counterpart required more than 10 min.