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The Paleoproterozoic Gordon Lake and Bar River formations, Huronian Supergroup, contain a variety of sedimentary structures in the Flack Lake area of Ontario, Canada, that have been considered of debatable origin. We identify these structures as microbially induced sedimentary structures (MISS). The preserved MISS are related to microbial mat destruction and decay, and include sand cracks, mat chips, remnant gas domes, pyrite patches, and iron laminae. A biological origin for the fossil structures is supported by their similarities to modern and ancient documented examples of MISS, the sand-dominated nature of the substrate in which they are preserved, and key microtextures identified in thin section. Microtextures include curled, frayed and layered mat chips, carbonaceous laminae, oriented grains, and concentrated heavy minerals. On outcrop scale, the presence of desiccation cracks, flaser and lenticular bedding, and ripples in association with the types of MISS identified in the Gordon Lake Formation support the interpretation of a tidal flat depositional environment. The Gordon Lake Formation contains a greater number and diversity of MISS than the overlying Bar River Formation, as a result of lower energy deposition in the former. The quartz arenite of the Bar River Formation contains fine-grained to pebbly granulestone characterized mainly by tangential and planar cross beds, which is consistent with a tidal channel or sand shoal setting. Although fossil evidence of life is rare in the rocks of the Huronian Supergroup, identification of MISS in the Flack Lake area provides a significant and convincing indication of microbial colonization at the time of deposition.