The ca. 3 Ga Farrel Quartzite (FQ, Western Australia) contains possible organic microfossils of unusual spindle-like morphology that are surprisingly large and complex, preserved along with spheroids. The unusual nature of the possible fossils, coupled with their antiquity, makes their interpretation as biogenic difficult and debatable. Here, we report 32 in situ carbon isotopic analyses of 15 individual FQ specimens. The spheroids and the spindle-like forms have a weighted mean δ13C value of –37‰, an isotopic composition that is quite consistent with a biogenic origin. Both the spheroids and the spindle-like structures are isotopically distinct from the background organic matter in the same thin section (weighted mean δ13C value of –33‰), which shows that the preserved microstructures are not pseudofossils formed from physical reprocessing of the bulk sedimentary organic material. When considered along with published morphological and chemical studies, these results indicate that the FQ microstructures are bona fide microfossils, and support the interpretation that the spindles were planktonic. Our results also provide metabolic constraints that imply most of these preserved microorganisms were autotrophic. The existence of similar spindles in the ca. 3.4 Ga Strelley Pool Formation of Australia and the ca. 3.4 Ga Onverwacht Group of South Africa suggests that the spindle-containing microbiota may be one of the oldest, morphologically preserved examples of life. If this is the case, then the FQ structures represent the remains of a cosmopolitan biological experiment that appears to have lasted for several hundred million years, starting in the Paleoarchean.