A sandstone from the ca. 3400 Ma Strelley Pool Formation of Western Australia contains pristine micron-sized pyrite intimately associated with organic material coating framework quartz grains. A synsedimentary to early diagenetic origin for this pyrite is indicated by its occurrence in black, bedded sandstone at the base of the formation, and in reworked black clasts higher up in the formation. High-resolution multiple sulfur isotope analysis (32S, 33S, 34S) using secondary ion mass spectrometry (NanoSIMS and large-radius ion microprobe) reveals δ34SVCDT (Vienna Canyon Diablo troilite) values between ∼–12‰ and +6‰, and Δ33S values between –1.65‰ and +1.43‰, from pyrite grains within a single thin section. A large spread of δ34S values over only 5–10 μm, together with the spatial association of pyrite with carbon and nitrogen, indicates biological processing of sulfur. The presence of both +Δ33S and –Δ33S signals overprinted by significant mass-dependent δ34S fractionation in this pyrite population indicates for the first time that both microbial sulfate reduction of aqueous sulfate (–Δ33S) and microbial disproportionation of elemental sulfur (+Δ33S) were co-occurring in an open-marine, sedimentary hosted ecosystem in the Paleoarchean.