The colonization of emergent continental landmass by microbial life was an evolutionary step of paramount importance in Earth history. Here we report direct fossil evidence for life on land 3,220 million years ago (Ma) in the form of terrestrial microbial mats draping fluvial conglomerates and gravelly sandstones of the Moodies Group, South Africa. Combined field, petrographic, carbon isotope and Raman spectroscopic analyses confirm the synsedimentary origin and biogenicity of these unique fossil mats as well as their fluvial habitat. The carbon isotope compositions of organic matter (δ13Corg) from these mats define a narrow range centred on -21‰, in contrast to fossil mats of marine origin from nearby tidal deposits that show δ13Corg values as low as -34‰. Bulk nitrogen isotope compositions (2 < δ15N < 5‰) are also significantly different from their marine counterparts (0 < δ15N < 3‰), which we interpret as reflecting denitrification in the terrestrial habitat, possibly of an atmospheric source of nitrate. Our results support the antiquity of a thriving terrestrial biosphere during the Palaeoarchaean and suggest that a complex and microbially driven redox landscape existed during the deposition of the Moodies Group, with distinct biogeochemical cycling occurring on land by 3,220 Ma.