Fluviolacustrine sediments of the 2.75 Ga Hardey Formation (Fortescue Group, Australia) preserve pendant columnar structures with stromatolitic lamination within synsedimentary cavities. The millimeter-sized, finger-like columns strongly resemble microbialites from modern basaltic caves and indicate the likely presence of microbial biofilms. The ancient microbial columns are preserved by chert and locally occur as reworked clasts, indicating a near-depositional age for the structures. Sulfur isotopic analysis of pyrite in the columns and adjacent carbonaceous matrix yields δ34SCDT (CDT—Canyon Diablo troilite) values between −8.5‰ and +19‰, showing significant fractionation characteristic of biological cycling of sulfur. Organic matter in cavity ceilings and shale matrix has δ13CPDB (PDB—Peedee belemnite) values between −55‰ and −43‰, suggesting the presence of methanotrophs. Our results suggest that 2.75 Ga terrestrial environments supported a microbial ecosystem, including microbes that inhabited synsedimentary hollows, extending the fossil record of coelobionts by ~1.5 b.y. Subsurface cavities represent a new habitable microenvironment for early life on Earth, and an analogue for ancient life on Mars.