Fluorescent D-amino acids (FDAAs) are efficiently incorporated into the peptidoglycans (PGs) of diverse bacterial species at the sites of PG biosynthesis, allowing specific and covalent probing of bacterial growth with minimal perturbation. Here we provide a protocol for the synthesis of four FDAAs emitting light in blue (HCC-amino-D-alanine, HADA), green (NBD-amino-D-alanine, NADA, and fluorescein-D-lysine, FDL) or red (TAMRA-D-lysine, TDL) and for their use in PG labeling of live bacteria. Our modular synthesis protocol gives easy access to a library of different FDAAs made with commercially available fluorophores and diamino acid starting materials. Molecules can be synthesized in a typical chemistry laboratory in 2-3 d using standard chemical transformations. The simple labeling procedure involves the addition of the FDAAs to a bacterial sample for the desired labeling duration and stopping further label incorporation by fixing the cells with cold 70% (vol/vol) ethanol or by washing away excess dye. We discuss several scenarios for the use of these labels in fluorescence microscopy applications, including short or long labeling durations, and the combination of different labels in pure culture (e.g., for 'virtual time-lapse' microscopy) or in situ labeling of complex environmental samples. Depending on the experiment, FDAA labeling can take as little as 30 s for a rapidly growing species such as Escherichia coli.