One of the key challenges in chemistry is to break and form bonds selectively in complex organic molecules that possess a range of different functional groups. To do this at the single-molecule level not only provides an opportunity to create custom nanoscale devices, but offers opportunities for the in-depth study of how the molecular electronic structure changes in individual reactions. Here we use a scanning tunnelling microscope (STM) to induce a sequence of targeted bond dissociation and formation steps in single thiol-based π-conjugated molecules adsorbed on a NiAl(110) surface. Furthermore, the electronic resonances of the resulting species were measured by spatially resolved electronic spectroscopy at each reaction step. Specifically, the STM was used to cleave individual acetyl groups and to form Au-S bonds by manipulating single Au atoms. A detailed understanding of the Au-S bond and its non-local influence is fundamentally important for determining the electron transport in thiol-based molecular junction.