Cholesterol plays an essential role in eukaryotic life as a structural building block of cell membranes and for signalling^1-2, and it is the precursor of vital biomolecules including bile acids or vitamin D³. While the mechanisms of cholesterol homeostasis have been largely investigated but the role of microbiome-related functions on cholesterol metabolism are still poorly understood. The gastro-intestinal tract hosts millions of bacteria, viruses, archaea, parasites and fungi that influence the metabolic abilities of their host. Many of these microbes generate metabolites that the enzymes from the host are not capable of producing⁴. In most individuals, much of cholesterol in the gut undergoes microbial conversion to its major metabolite in faeces, coprostanol. Coprostanol, a 5β-stanol, is uniquely formed through microbial saturation of its precursor Δ5 - sterol cholesterol by specific bacteria present in the gut of mammals⁵, and it's generation depends primarily on the diet, the endogenous cholesterol biosynthesis and the efficiency of the gut microbial action, which produces a specific chemical signature^6-7. Coprostanol, is the result of the conversion of dietary and de novo synthesized cholesterol⁶. This microbial conversion occurs in most humans, and prevents the reabsorption of cholesterol in the colon, which can be an advantage against cholesterol-related cardiovascular diseases⁴. Nevertheless, the microbial actors behind this conversion are yet to be elucidated, and only a few cholesterol reducing bacteria, mainly Bacteroides and Eubacterium, have proven to be able to convert cholesterol into coprostanol in vitro^8-9. Further, the genes or enzymes involved in this conversion remain poorly investigated. Once excreted, coprostanol is believed to remain mostly intact during early diagenesis, which together with the characteristic distribution of coprostanol and its homologues according to their origin, makes them ideal candidates to serve as faecal biomarkers for source assignment⁵. Recent studies have provided a great deal of information regarding the differences between animal faecal fingerprints. However, little is known about the individual variations and their relationships with the microbiota, as well as the preservation and early diagenesis of coprostanol in the fossil record. In order to assess the origin, preservation and early diagenesis of coprostanol, we present GC-MS data from faecal samples in different stages of maturation, including samples collected from healthy individuals with different diets and lifestyles, 1000 years-old mummified gut tissues and 50k years-old putative Neanderthal coprolites. We also aim to identify the main actors of this microbial conversion using a combined multiomic approach. In this study, we show a high diversity in faecal lipid contents, which can be related to inter-variation of gut microbiome. Our results highlight the complexity of 5β-stanol production in the gut and its application as a biomarker for source assignment. The application of a multiomic approach sheds light on the metabolism of cholesterol in the gut and the bacteria in the gut community carrying this conversion. A better understanding of the origin, preservation and diagenesis of coprostanol and its diagenetic products (coprostane) can open a new window onto comparative studies of contemporary and ancient microbiomes.