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Abstract We apply a new approach for the δ¹³C analysis of single organic‐walled microfossils (OWM) to three sites in the Appalachian Basin of New York (AB) that span the Late Devonian Biotic Crisis (LDBC). Our data provide new insights into the nature of the Frasnian–Famennian carbon cycle in the AB and also provide possible constraints on the paleoecology of enigmatic OWM ubiquitous in Paleozoic shale successions. The carbon isotope compositions of OWM are consistent with normal marine organic matter of autochthonous origins and range from −32 to −17‰, but average −25‰ across all samples and are consistently¹³C‐enriched compared to bulk sediments (δ¹³C_bulk) by ~0–10‰. We observe no difference between the δ¹³C_OWMof leiospheres (smooth‐walled) and acanthomorphic (spinose) acritarch OWM, indicating that our data are driven by ecological rather than taxonomic signals. We hypothesize that the offset between δ¹³C_OWMand δ¹³C_bulkis in part due to a large δ¹³C gradient in the AB water column where OWM utilized relatively¹³C‐enriched dissolved inorganic carbon near the surface. Thus, the organisms producing the balance of the total organic carbon were assimilating¹³C‐depleted C sources, including but not limited to respired organic carbon or byproducts of fermentation. We also observe a systematic decrease in both δ¹³C_OWMand δ¹³C_bulkof 3‰ from shoreward to open‐ocean facies that may reflect the effect of¹³C‐enriched dissolved inorganic carbon (DIC) derived from riverine sources in the relatively enclosed AB. The hypothesized steep carbon isotope gradient in the AB could be due to a strong biological pump; this in turn may have contributed to low oxygen bottom water conditions during the LDBC. This is the first time single‐microfossil δ¹³C_organalyses of eukaryotes have been directly compared to bulk δ¹³C_orgin the deep‐time fossil record.