The fault-controlled Nibao Carlin-type gold deposit, together with the strata-bound Shuiyindong deposit, comprise a significant amount of the disseminated gold deposits in southwestern Guizhou Province, China. Five main types and two sub-types of pyrite at the Nibao deposit (Py1a/Py1b, Py2, Py3, Py4, Py5) were distinguished based on detailed mineralogical work. Py1, Py2 and Py3 are Au-poor, whereas Py4 and Py5 are Au-rich, corresponding to a sedimentary and hydrothermal origin, respectively. Through systematic in situ analyses of NanoSIMS sulfur isotopes, the framboid pyrite Py1a with negative δ³⁴S values (− 53.3 to − 14.9‰) from the Nibao deposit were found to originate from bacterial sulfate reduction (BSR) processes in an open and sulfate-sufficient condition while the superheavy pyrite Py1b (73.7–114.8‰) is probably due to the potential influence of closed-system Rayleigh fractionation or the lack of preservation of deep-sea sediments. Data of Py2 and Py3 plot within the area of S isotope compositions from biogenic and abiogenic sulfate reduction. In view of few coeval magmatic rocks in the mining district, the near zero δ³⁴S values of the Au-rich pyrites (Py4 and Py5) may discount the potential involvement of magmatic but metamorphic or sedimentary origin. LA-ICP-MS and TEM work show that Au in ore-related pyrite is present as both nanoparticles and structurally bound. LA-ICP-MS analyses show that the Au-rich pyrite also contains higher As, Cu, Sb, Tl and S than other types of pyrite, which inferred a distal manifestation of deep hydrothermal mineralization systems.