Multiple sulfur isotope variability in Archean sedimentary rocks provides constraints on the composition of the Earth’s earliest atmosphere. The magnitude and sign of mass-independent anomalies reflect not only atmospheric processes, but also transformations due to the Archean marine sulfur cycle prior to preservation into sedimentary pyrite. The processes affecting the Archean marine sulfur cycle and the role of microbial or abiotic redox reactions during pyrite formation remain unclear. Here we combine iron (Fe) and multiple sulfur (S) isotope data in individual pyrite grains with petrographic information and a one-dimensional reactive transport model, to investigate the sources of Fe and S in pyrite formed in a Paleoarchean sedimentary basin. Pyrites were selected from mudstones, sandstones and chert obtained from a drill core in the ca. 3.2 Ga Mapepe and Mendon Formations of the Fig Tree and Onverwacht Groups, respectively, in the Barberton Greenstone Belt, Kaapvaal Craton, South Africa. Pyrite textures and δ56Fe distinguish early-diagenetic pyrite formed with pore-water ferrous iron (disseminated grains with average δ56Fepyrite = 0‰) from late-diagenetic pyrite formed through sulfidation of iron oxide minerals (layered and aggregate forms with average δ56Fepyrite = + 1‰). Mass dependent S isotope variability in pyrite was small (δ34Spyrite ranged from − 1.1 to + 3.3‰) with a correspondingly minor spread in Δ33Spyrite (ranging from + 0.3 to + 2.1‰) and Δ36Spyrite (ranging from − 3.08 to + 0.27‰) that indicates a lack of post-depositional re-working with other distinct sulfur sources. Our combined Fe and S isotope data are most readily explained with pyrite sulfide derived from microbial-reworking of solid elemental S. Iron oxide minerals were necessary to buffer sulfide concentrations and provide favorable conditions for microbial sulfur disproportionation to proceed. The lack of a negative Δ33S signal indicates that pyrite from relatively deep marine diagenetic environments only partially records the products of atmospheric photolysis, consistent with low sulfate concentrations in the Paleoarchean ocean.