The subcellular organization of biological molecules is a critical determinant of many bacterial processes, including growth, replication of the genome, and division, yet the details of many mechanisms that control intracellular organization remain unknown. Decoding this information will impact the field of bacterial physiology and can provide insight into eukaryotic biology, including related processes in mitochondria and chloroplasts. Small molecule probes provide unique advantages in studying these mechanisms and manipulating the organization of biomolecules in live bacterial cells. In this review, we describe small molecules that are available for investigating subcellular organization in bacteria, specifically targeting FtsZ, MreB, peptidoglycan, and lipid bilayers. We discuss how these probes have been used to study microbiological questions and conclude by providing suggestions about important areas in which chemical-biological approaches will have a revolutionary impact on the study of bacterial physiology.