Esa1 (essential Sas2-related acetyltransferase 1) and Tip60 (HIV-1 TAT-interactive protein, 60 kDa) are key members of the MYST family of histone acetyltransferases (HATs) and play important functions in many cellular processes. In this work, we designed, synthesized and evaluated a series of substrate-based analogs for the inhibition of Esa1 and Tip60. The structures of these analogs feature that coenzyme A is covalently linked to the side chain amino group of the acetyl lysine residues in the histone peptide substrates. These bisubstrate analogs exhibit stronger potency in the inhibition of Esa1 and Tip60 compared to the small molecules curcumin and anacardic acid. In particular, H4K16CoA was tested as one of the most potent inhibitors for both Esa1 and Tip60. These substrate-based analog inhibitors will be useful mechanistic tools for analyzing biochemical mechanisms of Esa1 and Tip60, defining their functional roles in particular biological pathways, and facilitating protein crystallization and structural determination.