The oxygen isotopic composition of conodont apatite from two Frasnian-Famennian boundary sections was measured in order to reconstruct variations in marine paleotemperatures during the late Frasnian mass-extinction event. The measured conodont apatite δ18O values reveal two positive excursions with maximum amplitudes of +1‰ to +1.5‰ that parallel positive excursions in the carbonate carbon isotopic composition. The +3‰ excursions in carbonate δ13C have been interpreted as consequences of enhanced organic carbon burial rate resulting in a decrease in atmospheric CO2 concentration. Climatic cooling as a potential consequence of lower atmospheric CO2 concentration is confirmed by the conodont apatite δ18O records, which translate into cooling of low-latitude surface waters by 5–7 °C. Repeated cooling of the low latitudes during the late Frasnian had a severe impact on the tropical shallow-water faunas that were probably adapted to warm surface-water temperatures and severely affected during the late Frasnian crisis. These prominent variations in ocean-water temperature were stressful to the tropical shallow-water fauna and potentially culminated in low origination rates of new species, one of the major factors of the decline in diversity during the latest Frasnian.