An approach for the identification of the origin of iron sulfides formation on iron surfaces using Time-of-Flight Secondary Ions Mass Spectrometry (ToF-SIMS) is reported here in. Two different sulfidation processes of iron surfaces are presented (both at room temperature): the first abiotic procedure was carried out using chronoamperometry (E= -0.8 V/SCE) of pure iron in 10 mM of Na2S, 9H(2)O (pH= 11) while the second biotic procedure was accomplished by immersing a pure iron sample in a medium containing HS- ions produced by a sulfate-reducing bacterium (SRB) of the genus Desulfovibrio at an open circuit potential (OCP). A surface analytical method using Time-of-Flight Secondary Ions Mass Spectrometry (ToF-SIMS) coupled with ToF-SIMS spectra peak-fitting and data processing was developed to calculate an accurate sulfur isotopic fractionation delta 34S(V-CDT) on iron sulfide layers formed on iron surfaces, thus allowing to discriminate between abiotically versus biotically generated sulfides. This approach contributes to a better understanding of iron surface interactions with sulfur containing environmental species of abiotic or biotic origin.