Internal textures, multiple sulfur isotopic compositions, and contents of gold, selenium, and molybdenum of ore-related pyrites from a Neoarchean carbonate-facies BIF-hosted gold deposit in Quadrilátero Ferrífero were investigated to elucidate the source(s) of sulfur. Sodium hypochlorite etching and BSE imaging revealed pervasive zoning in pyrites. Five different growth zones (Py1a, Py1b, Py2, Py3, and Py4) and six types of zoning (Type A–Type F) were identified. Two pyrite generations were distinguished in ores: G1 (Py1a and Py1b) and G2 (Py2, Py3, Py4). Both G1 and G2 have positive Δ33S, but the magnitudes of G1 are higher. The G1-relevant fluids can be shallow-sourced, whereas the G2-related auriferous fluids are most likely deep-sourced metamorphic fluids derived from devolatilization of the lower succession of the Nova Lima Group (metavolcanics and metasedimentary rocks) during metamorphism, with fluid–rock interactions during fluid ascent and at the depositional site. The negative δ34S, higher selenium contents, and carbonates inclusions of Py1a contrast with the positive δ34S, lower selenium contents, and carbonaceous material inclusions of Py2, Py3, and Py4. The Lamego system possibly started with shallow-sourced oxidized and low-gold fluids (Py1a), closely followed by mixing in of deep-seated reduced auriferous fluids (Py2, Py3, Py4) initiated by tectonic activities. The oscillatory zoning of Py2 with the highest gold contents consists of alternating gold-rich and gold-poor laminae, indicating that fault-valve activity is a trigger of gold deposition. The sulfidation of siderite in BIF (desulfidation of auriferous fluids) related to the formation of ore-related pyrites also contributed to gold precipitation.