Recent developments on fluorescent proteins and microscopy techniques have allowed the probing of single molecules in a living bacterial cell with high specificity, millisecond time resolution, and nanometer spatial precision. Recording movies and analyzing dynamics of individual macromolecules have brought new insights into the mechanisms of many processes in molecular biology, such as DNA-protein interactions, gene regulation, transcription, translation, and replication, among others. Here we review the key methods of single-molecule detection and highlight numerous examples to illustrate how these experiments are contributing to the quantitative understanding of the fundamental processes in a living cell.