Infrared attenuated total reflection (IR-ATR) spectroscopy was applied to analyze carbonates in sediment samples collected at a cold seep site in Mississippi Canyon 118, Gulf of Mexico. Previously, these samples were grouped into low, moderate, and high microbial activity based on sulfate and methane down-core profiles. We have hypothesized that within these groups, IR-ATR spectroscopy could differentiate between cold seep derived microbially mediated authigenic carbonates found in high and moderate microbially active sediments, and biogenically produced carbonates representative for low microbially active sediments. Within the respective IR spectroscopic absorption profiles, the ν 3 antisymmetric carbonate stretching vibration was identified as a suitable indicator within these diverse geochemical groupings. Low microbial activity cores revealed absorption profiles that are significantly different from those of high and moderate microbial activity cores. To verify the IR-ATR results, the obtained spectral profiles were compared to stable isotopic values of in situ bulk carbonate signals. It is shown that for carbonates depleted in δ 13C (negative values), the IR ν 3 profiles are uniquely different. To semi-quantify this method, the obtained IR profiles were utilized for deriving an IR indicator ( F) for establishing an analytical model suitable for the identification of cold seep derived authigenic carbonate. The sediment samples characterized by cold seep derived authigenic carbonates have an average indicator F value of 104.3 ± 15.8, whereas biogenic carbonates show F values of 53.5 ± 11.4. The obtained results demonstrate that IR-ATR spectroscopy may be applied as a shipboard and potentially in situ research tool for rapid and cost-effective characterization of carbonate formations in cold seep ecosystems.