Sulphur isotope data from early Archaean rocks suggest that microbes with metabolisms based on sulphur existed almost 3.5 billion years ago, leading to suggestions that the earliest microbial ecosystems were sulphur-based. However, morphological evidence for these sulphur-metabolizing bacteria has been elusive. Here we report the presence of microstructures from the 3.4-billion-year-old Strelley Pool Formation in Western Australia that are associated with micrometre-sized pyrite crystals. The microstructures we identify exhibit indicators of biological affinity, including hollow cell lumens, carbonaceous cell walls enriched in nitrogen, taphonomic degradation, organization into chains and clusters, and δ13 C values of -33 to -46% Vienna PeeDee Belemnite (VPDB). We therefore identify them as microfossils of spheroidal and ellipsoidal cells and tubular sheaths demonstrating the organization of multiple cells. The associated pyrite crystals have Δ33 S values between -1.65 and +1.43% and Δ34 S values ranging from -12 to +6% Vienna Canyon Diablo Troilite (VCDT) 5. We interpret the pyrite crystals as the metabolic by-products of these cells, which would have employed sulphate-reduction and sulphur-disproportionation pathways. These microfossils are about 200 million years older than previously described microfossils from Palaeoarchaean siliciclastic environments.