Biocompatible gold nanoparticles (AuNPs) are used in wound healing due to their radical scavenging activity. They shorten wound healing time by, for example, improving re-epithelialization and promoting the formation of new connective tissue. Another approach that promotes wound healing through cell proliferation while inhibiting bacterial growth is an acidic microenvironment, which can be achieved with acid-forming buffers. Accordingly, a combination of these two approaches appears promising and is the focus of the present study. Here, 18 nm and 56 nm gold NP (Au) were prepared with Turkevich reduction synthesis using design-of-experiments methodology, and the influence of pH and ionic strength on their behaviour was investigated. The citrate buffer had a pronounced effect on the stability of AuNPs due to the more complex intermolecular interactions, which was also confirmed by the changes in optical properties. In contrast, AuNPs dispersed in lactate and phosphate buffer were stable at therapeutically relevant ionic strength, regardless of their size. Simulation of the local pH distribution near the particle surface also showed a steep pH gradient for particles smaller than 100 nm. This suggests that the healing potential is further enhanced by a more acidic environment at the particle surface, making this strategy a promising approach.