Lysine acetylation is an evolutionarily conserved protein modification that regulates different cellular pathways. The acetyl moiety can be transferred to the lysine side chain in two ways, enzymatically by lysine acetyltransferases and non-enzymatic. Usually, acetyl coenzyme A is the donor agent, although acetyl phosphate is the main regulator of acetylation in bacteria. The removal of the acetyl group occurs exclusively enzymatically. In prokaryotes, lysine acetyltransferases are grouped by a core structural domain architecture in the protein superfamily Gcn5-related N-acetyltransferase (GNAT). With different implications, these enzymes can acetylate the amino group of small molecules, metabolites, peptides, and proteins. As well as mitochondria, chemical acetylation has also been demonstrated as a global regulatory mechanism in bacteria. This review presents current knowledge of acetylation mechanisms and functional implications in bacteria metabolism. Additionally, the advances in mass spectrometry for studying this PTM, including relative quantification and stoichiometry quantification, and how these methods have allowed researchers to elucidate the biological significance in bacteria are reviewed.