<p>Abundant graphitized organic matter within 3.7-billion-year-old sediments in the Isua Supracrustal Belt comprises the oldest remnants of life. This organic matter could have provided a favorable substrate for anaerobically respiring microbes, though their existence in the early Archean remains uncertain. Here we assess whether anaerobic respiration, linked to reduction of iron and sulfur, operated within these ancient sediments. We analyzed carbon and sulfur isotope data from pelagic and turbiditic sedimentary rocks, sampled from a rock core, and used petrography and iron concentrations to provide geological context. Carbon isotopic compositions indicate respiration of organic compounds, with lighter values associated with iron-rich samples, consistent with respiration coupled to iron reduction. Sulfide grains in pelagic layers have isotopic compositions consistent with reduction of atmospherically produced elemental sulfur, possibly with minor contributions from sulfate reduction during sedimentary hiatuses. These results suggest that early Archean ecosystems were sustained by multiple, interacting microbial metabolisms.</p>