Paleoproterozioc (ca. 1.9 Ga) stromatolites from the Biwabik Iron-Formation (Minnesota, United States) contain fossil forms which in the literature have been attributed to ancient microbes including iron-oxidising bacteria and cyanobacteria. To search for valence state fossils, we measured Fe³⁺/ΣFe (ΣFe = Fe²⁺ + Fe³⁺) transverse to the laminae of a Biwabik, probably biogenic stromatolite and compared the results with a scan across the enclosing, likely abiotic sedimentary rock (or interstromatolite fill). To obtain Fe³⁺/ΣFe and information about the site symmetry and crystal field strength, we used L₃ (2p_3/2 → 3d) soft X-ray absorption spectroscopy and interpreted the measurements using our calculated spectra. We found that Fe³⁺/ΣFe is approximately constant at 0.3 within the stromatolite, increases steeply to around 0.6 in the region of the stromatolite-fill boundary, and then reduces significantly in the interstromatolite fill. The crystal field strength for octahedral Fe³⁺ increased and became more irregular as the scan moved from the stromatolite into the fill. Our results tentatively suggest that a combination of ancient biological and later diagenetic processes can produce steep gradients in Fe³⁺/ΣFe and alterations in the crystal field strength across a probable abiotic-biotic boundary. To describe the L₃ absorption spectra in the stromatolite-fill region, it was necessary to include a significant Fe³⁺ tetrahedral component, which suggests the presence of magnetite. At other positions, fits using only octahedral Fe²⁺ and Fe³⁺ theoretical curves satisfactorily reproduce the spectra. The search for a possible new type of spatial biosignature in the valence state record for ancient stromatolites might help differentiate between the biogenic stromatolites and abiogenic stromatolite-like structures and could even be relevant to the search for fossil evidence of life in extraterrestrial rocks.