Lipids play important biological roles and their metabolism is closely related to the development and progression of a variety of diseases. Lipid analysis is of great importance for research on disease mechanisms, biomarker discovery, diagnosis and treatment, and drug research. Lipidomics systematically analyze the lipids and molecules that interact with lipids in biological samples. This review introduces the biological roles of lipids and the analytical methods regarding lipidomics in microbiology, and offers a future view of lipidomics in microbiology.