Ovothiol A is a 5-thiohistidine derivative biosynthesized by a broad range of prokaryotic and eukaryotic organisms. Its redox-active mercaptoimidazole side chain is believed to protect cells from oxidative stress. The three enzymes that produce ovothiol A from histidine, cysteine, and S-adenosyl methionine have been identified and characterized. In contrast, no enzymes are known that produce other 5-thiohistidine derivatives. Here, we describe a small family of acetyl-coenzyme A-dependent transferases that produce N-acetyl-5-thiohistidine. The discovery of these enzymes from Proteiniphilum saccharofermentans and related Bacteroidota provides evidence that the 5-thiohistidine class may be structurally and functionally more diverse than previously thought.