The naphthalene ring is a moiety featured by many bioactive agents. Dihydroxynaphthalenes are readily available substances but their use for the synthesis of a series of compounds results in limited chemical diversity due to the similar reactivity of the phenolic hydroxyl groups. Herein, we describe the use of regioselective lipases in acylation and hydrolysis reactions to synthesize 5 different regioisomeric acetoxyhydroxynaphthalenes on a 91-122 mg scale with moderate to good yields (50-78%) from 1,3-, 1,6- and 1,7-dihydroxynaphthalenes and their corresponding diacetates. Reactions were optimised through medium engineering, thus providing information on the impact of different organic solvents on conversion and selectivity. Additionally, computational studies using molecular dynamic simulations were performed and suggested a correlation between the regiopreference displayed by lipase CAL-B in hydrolytic reactions and a distance ≤3.2 Å between the most reactive carbonyl group and the Ser-105 residue on the catalytic site. The herein described enzymatic methods may allow for introducing two different moieties at the naphthalene ring through a protection-deprotection strategy, thus allowing for better exploitation of the chemical space.