Four Deep Eutectic Solvents (DES) based on choline chloride (ChCl) and different hydrogen bond donors (1,4-butanediol, glycerol, isosorbide, and urea) were tested as reaction medium for the immobilized Candida antarctica B (iCALB) lipase-catalyzed esterification of fatty acids with n-butanol. Although the strong hydrogen bonds between DES components were expected to lower their reactivity, all except urea, competed in the esterification reaction. To study the effect of acyl group donor and water, different chain length of saturated fatty acids (ranging from 6:0 to 18:0) and water content (ranging from 0, 1, 2 or 3 molar ratio of water/ChCl) were tested in the lipase-catalyzed esterification with n-butanol in ChCl:urea (1:2, molar ratio). When the incubation was performed in the absence of water, the shorter the fatty acid chain length, the higher the conversion. Upon water addition, a drastic increase in the esterification rate of all the fatty acids was observed and, conversely to what occurred in the anhydrous media, the longer the fatty acid chain length, the higher the conversion. Results suggest that water concentration in the DES medium possesses a multifactorial effect, as it influences the enzyme activity, the solvent viscosity, the DES structure, and the interaction between DES components, substrates, and enzymes, thus determining the efficiency and yield of the esterification reaction.