Abstract Filament-shaped titanite microtextures in early Archaean (c. 3.47 Ga) metabasalts from the Barberton Greenstone Belt of South Africa have been argued to represent Earth’s oldest microbial trace fossils, but petrological data on the environmental conditions that led to the formation of these microtextures are sparse. We present here new metamorphic constraints on the equilibrium mineral assemblage containing the titanite microtextures, together with Fe speciation redox variations in the chlorite matrix surrounding the microtextures and morphospace analysis of the microtextures to test their biogenicity. Thermodynamic phase diagram modelling using a calculated rock microdomain composition indicated that the titanite mineral assemblage is stable at temperature conditions of T =240–360°C. The high-resolution quantitative mapping results combined with micro-X-ray absorption near-edge spectroscopy at the Fe K -edge in chlorite revealed that the titanite microtextures are located in low-temperature, high XFe 3+ chlorite bands, veins and microdomains, supporting an origin as abiotic, retrograde mineral cooling textures. These new data, combined with a late c. 2.9 Ga U–Pb date for the titanite, are incompatible with the existing biological model, which invokes microbial micro-tunnel formation followed by titanite ‘infilling’ and preservation in the early Archaean sub-seafloor. The continuum of titanite morphologies reported herein indicates that titanite morphology cannot be used as reliable evidence in support of a biogenic origin in these metavolcanic rocks. It is proposed that the titanite microtextures of purported biogenic origin from other greenstone belts, such as the Pilbara Craton of Western Australia, also deserve to be scrutinized by high-resolution petrological investigations. Supplementary material: Representative electron microprobe analyses of retrograde minerals and µXANES spectra and corresponding fitted pre-edge peaks for all spot analyses are available at https://doi.org/10.6084/m9.figshare.c.3498465