Orogenic gold deposits are commonly associated with a small alteration and geochemical footprint that often hinders the exploration process. In the Granites-Tanami Gold Province; however, we demonstrate that sulfur isotope analyses represent a powerful exploration tool for orogenic gold mineralization. The Granites-Tanami Gold Province, situated in the Northern Territory (Australia), hosts numerous gold occurrences including the world-class Callie deposit (c. 14.2 Moz Au). In this paper, we use sulfur isotopes to: 1) characterize the δ34S signature of the sulfides associated with gold mineralization in order to assist with exploration in the Granites-Tanami Gold Province, and 2) monitor the temporal fluid evolution of the Callie deposit. We present in-situ sulfur isotope analyses of sulfides associated to gold mineralization, diagenetic sulfides, and sulfides associated to a barren, post-mineralization hydrothermal event. Our results show that the sulfides associated with mineralization yield δ34S values that range from +4‰ to +11.5‰ (n = 114), the diagenetic sulfides yield δ34S values that range from +0.0‰ to +1.7‰ (n = 41) and the post-mineralization sulfides yield δ34S values that range from +2.3‰ to +3.3‰ (n = 20). The range of δ34S values obtained from mineralized samples at the Callie deposit coincides with δ34S values reported in sulfides associated to other orogenic gold deposits of the Granites-Tanami Gold Province. The mineralization-related δ34S signature in the Granites-Tanami Gold Province is distinct from both diagenetic and post-mineralization sulfides providing a unique isotopic signature for the sulfides associated to gold mineralization. Therefore, we propose that the application of routine sulfur isotope analysis may represent a valuable tool for mineral exploration in the area. We also report a temporal shift toward lighter δ34S values from various sulfides associated to the stratabound mineralization at the Callie deposit. We interpret the shift in δ34S values as the expression of a progressive increase in the oxygen fugacity of the ore fluid resulting from progressive alteration of the host-rock.