Stromatolites of the ∼3.5 billion-year-old Dresser Formation (Pilbara Craton, Western Australia) are considered to be some of Earth’s earliest convincing evidence of life. However, uniquely biogenic interpretations based on surface outcrops are precluded by weathering, which has altered primary mineralogy and inhibited the preservation of microbial remains. Here, we report on exceptionally preserved, strongly sulfidized stromatolites obtained by diamond drilling from below the weathering profile. These stromatolites lie within undeformed hydrothermal-sedimentary strata and show textural features that are indicative of biogenic origins, including upward-broadening and/or upward-branching digitate forms, wavy to wrinkly laminae, and finely laminated columns that show a thickening of laminae over flexure crests. High-resolution textural, mineralogical, and chemical analysis reveals that the stromatolites are dominated by petrographically earliest, nano-porous pyrite that contains thermally mature, N-bearing organic matter (OM). This nano-porous pyrite is consistent with a formation via sulfidization of an originally OM-dominated matrix. Evidence for its relationship with microbial communities are entombed OM strands and filaments, whose microtexture and chemistry are consistent with an origin as mineralized biofilm remains, and carbon isotope data of extracted OM (δ13C OM = −29.6‰ ± 0.3‰ VPDB [Vienna Peedee belemnite]), which lie within the range of biological matter. Collectively, our findings provide exceptional evidence for the biogenicity of some of Earth’s oldest stromatolites through preservation of OM, including microbial remains, by sulfidization.