Covering: up to August 2024Macrolides, the core skeletons of numerous marketed drugs and bioactive natural products, have garnered considerable scientific interest owing to their structural diversity and broad spectrum of pharmaceutical activities. The formation of intramolecular ester bonds is a critical biocatalytic step in constructing macrolide skeletons. Here, we summarised enzymatic ester bond formation strategies in fungal polyketide (PK)-type, nonribosomal peptide (NRP)-type, and PK-NRP hybrid-type macrolides. In PK-type macrolides, ester bond formation is commonly catalysed by a trans-acting thioesterase (TE) or a cis-acting TE domain during the product release process. In NRP-type and PK-NRP hybrid-type macrolides, the ester bond is usually introduced through condensation (C) domain-catalysed esterification during the elongation or product release step. Although the TE and C domains share similarities in their catalytic mechanism, using hydroxyl groups as nucleophiles in an intramolecular nucleophilic attack, they differ in terms of the hydroxyl origin, the timing of ester bond formation, and domain location. Furthermore, some TE domains are utilized as chemoenzymatic catalysts to construct macrolides with different ring sizes. A comparison of ester bond formation between fungi and bacteria is also discussed. Exploring the biosynthetic pathways of fungal macrolides, elucidating the diverse strategies employed in the formation of ester bonds, and understanding the application of enzymes/domains in chemoenzymatic synthesis hold promise for the discovery of new bioactive macrolides in the future.