The Archean sub-seafloor has been proposed as an environment for the emergence of life, with septate clusters of titanite microtextures in pillow lava rims argued to be the earliest traces of microbial microboring. Here we use nanoscale secondary ion mass spectrometry (NanoSIMS) to test possible geochemical traces of life in ca. 3.45 Ga pillow lavas of the Barberton Greenstone Belt, South Africa. Sulfide inclusions in the titanite microtextures record strongly negative sulfur isotope fractionations of δ34SVCDT –39.8‰ to –3.2‰ (VCDT—Vienna Canyon Diablo Troilite). These represent the largest range and most negative δ34S values so far reported from the Archean, and are consistent with an early biogenic origin for the sulfides. Extensive in situ elemental mapping did not find any organic linings associated with the microtextures, despite the high spatial resolution and sensitivity of the NanoSIMS. The absence of organic linings thus excludes a key line of evidence previously used to support the biogenicity of the microtextures. In contrast, in situ sulfur isotope analysis of basalt-hosted sulfides provides an alternative approach to investigating the existence and nature of an Archean subseafloor biosphere.