The GCN5-related N-acetyltransferase (GNAT) superfamily of proteins, widespread in eukaryotes and prokaryotes, can utilize acyl coenzyme A (acyl CoA) to acylate respective acceptor substrates and release both CoA and the acylated products. GNATs have been shown to be involved in multiple physiological events, including bacterial drug resistance, regulation of transcription, stress reaction, and metabolic flux, etc. In the last few years, the importance of GNATs has only emerged in eukaryotes, but bacterial GNATs, particularly those of pathogens, have only recently been explored. In this review, we summarize the main members, structures, inhibitors, and activators of proteins in the GNAT family. We focus on the roles of GNATs in bacteria, particularly Mycobacterium tuberculosis GNATs.