This study investigates the chemistry of the Au–thiol (Au–S) interface at the nanoscale using single-molecule flicker noise measurements. The research employs a mechanically controlled break junction technique to probe the conductance and analyze flicker noise for various aliphatic and aromatic thiol derivatives and thioethers. The findings demonstrate that flicker noise can unambiguously differentiate between stronger chemisorption (Au–SR) and weaker physisorption (Au–SRR') interactions. The results show that the gold rearrangement in chemisorbed Au–SR junctions resembles that of pure Au–Au metal contacts, independent of molecular structure. In contrast, thioethers form weaker physisorbed Au–SRR' bonds, with flicker noise indicating changes in the Au-anchoring interface but not Au–Au rearrangement. The study also explores the interfacial electric field-catalyzed ring-opening reaction of cyclic thioethers under mild conditions, which is otherwise difficult to achieve with harsh chemical conditions. Conductance measurements are complemented by NEGF transport calculations, highlighting the utility of flicker noise in monitoring chemical reactions at the single-molecule level. The research reveals that the nature of Au–S and Au–Au bonds is highly dynamic under different conditions, leading to inconsistent experimental observations. Flicker noise analysis provides a reliable method to probe interfacial reactions and interactions, distinguishing between chemisorption and physisorption. The study underscores the importance of understanding the Au–S interface for designing new synthesis techniques and developing systems with potential applications. The findings demonstrate that single-molecule conductance and flicker noise measurements can be used to tune and monitor chemical reactions at the single-molecule level.This study investigates the chemistry of the Au–thiol (Au–S) interface at the nanoscale using single-molecule flicker noise measurements. The research employs a mechanically controlled break junction technique to probe the conductance and analyze flicker noise for various aliphatic and aromatic thiol derivatives and thioethers. The findings demonstrate that flicker noise can unambiguously differentiate between stronger chemisorption (Au–SR) and weaker physisorption (Au–SRR') interactions. The results show that the gold rearrangement in chemisorbed Au–SR junctions resembles that of pure Au–Au metal contacts, independent of molecular structure. In contrast, thioethers form weaker physisorbed Au–SRR' bonds, with flicker noise indicating changes in the Au-anchoring interface but not Au–Au rearrangement. The study also explores the interfacial electric field-catalyzed ring-opening reaction of cyclic thioethers under mild conditions, which is otherwise difficult to achieve with harsh chemical conditions. Conductance measurements are complemented by NEGF transport calculations, highlighting the utility of flicker noise in monitoring chemical reactions at the single-molecule level. The research reveals that the nature of Au–S and Au–Au bonds is highly dynamic under different conditions, leading to inconsistent experimental observations. Flicker noise analysis provides a reliable method to probe interfacial reactions and interactions, distinguishing between chemisorption and physisorption. The study underscores the importance of understanding the Au–S interface for designing new synthesis techniques and developing systems with potential applications. The findings demonstrate that single-molecule conductance and flicker noise measurements can be used to tune and monitor chemical reactions at the single-molecule level.