Sterols are essential eukaryotic lipids that can be preserved as steroids in sedimentary rocks for billions of years. Because eukaryotes are the predominant modern day producers of these lipids, fossilized sterols are used as geological biomarkers for the presence of specific eukaryotes in ancient environments. Sterol lipids are also produced by a few bacteria, but the biosynthesis and function of sterols in bacteria are not as well-understood. In this study, we used a combination of bioinformatics and lipid analyses to identify bacterial sterol synthesis proteins. Our results indicate that bacteria have evolved distinct aspects of the sterol synthesis pathway independent of eukaryotes and show that exploring sterol physiology in bacteria can provide insight into this geologically relevant pathway.