The increasing bacterial resistance and negative impacts of the present antibacterial agents have led to the search for novel antibacterial agents. This study focuses on the influence of synthetic methods on the aggregation stability and antibacterial activity of gold nanoparticles (NPs) prepared by using sodium citrate as a reducing and capping agent againstStaphylococcus aureus(S. aureus). Gold NPs were synthesized using a simple and rapid sonochemical method and compared to gold NPs synthesized using a reduction method. The physicochemical features of gold NPs were characterized using UV-vis, XRD, TEM, and zeta potential, and the TEM results showed that the sonochemical method produced monodispersed spherical gold NPs with an average diameter of 18.5 nm, while the reduction method produced NPs with an average diameter of around 20 nm. The sonochemical method produced gold NPs with excellent stability (−48 mV) compared to the reduction method (−21 mV). The gold NPs with high stability also exhibited strong antibacterial activity againstS. aureuspresent in water, indicating their potential use in water purification processes to limit bacterial growth. The outcomes of this research are expected to significantly contribute to the creation of new drugs by paving the way for the development of novel strategies to combat pathogens using highly stable gold nanoparticles. These gold NPs, produced via the sonochemical method, have the potential to be employed as beneficial nanocompounds in the medical industry.