Metabolomics has become a powerful tool in chemical biology. Profiling the human sterolome has resulted in the discovery of noncanonical sterols, including oxysterols and meiosis-activating sterols. They are important to immune responses and development, and have been reviewed extensively. The triterpenoid metabolite fusidic acid has developed clinical relevance, and many steroidal metabolites from microbial sources possess varying bioactivities. Beyond the prospect of pharmacognostical agents, the profiling of minor metabolites can provide insight into an organism's biosynthesis and phylogeny, as well as inform drug discovery about infectious diseases. This review aims to highlight recent discoveries from detailed sterolomic profiling in microorganisms and their phylogenic and pharmacological implications.