The sponge biomarker hypothesis argues that 24-isopropylcholestanes preserved in Neoproterozoic-age rocks are “molecular fossils” left behind by marine sponges. Despite genetic and geologic support for this hypothesis, 24-isopropylcholestane has never been reported from a sponge body fossil. This lack of direct evidence regarding the source of sponge biomarkers through deep time leaves unanswered questions, such as whether their biosynthesis evolved once in sponges or multiple times across different lineages. In this study, we analyzed 10 sponge fossils from the Yale Peabody Museum of Natural History collections in pursuit of evidence of sterane biomarkers. We failed to recover 24-isopropylcholestane and instead found a near-identical sterane profile across all samples. This result indicates a combination of little to no sterane preservation in the fossils themselves, coupled with anthropogenic hydrocarbon contamination during their collection and storage. However, signals from bacterial biomarkers (hopanes) were more diverse across samples and consistent with a priori expectations, meaning that we cannot rule out the possibility that at least part of the hydrocarbon signal is syngenetic. We suggest that future attempts to extract biomarker hydrocarbons from sponge fossils be performed on freshly collected and specially prepared field samples. Despite the fact that demosponges or their ancestors still present the most likely source of Neoproterozoic 24-isopropylcholestanes, multiple evolutionary scenarios are consistent with current genetic and biomarker evidence: the “sponge biomarker” could represent an evolutionary novelty in demosponges, or a trait that evolved deeper in the animal tree. We therefore continue to affirm the validity of the sponge biomarker hypothesis but caution against using Neoproterozoic 24-isopropylcholestanes as a calibration point for sponges in molecular clocks. Instead, we recommend using it as a reference point for comparison, as scenarios where crowngroup demosponges radiate after the Neoproterozoic remain inconsistent with the geologic record.