A new efficient chemoenzymatic methodology for the production of 3-O-benzyl-sn-glycerol and 1, 2-O-dipalmitoyl-sn-glycerol has been developed. It starts from racemic 1-O- benzylglycerol and is based on the sequential enzymatic acylation––Mitsunobu inversion–– enzymatic hydrolysis, which has been performed without isolation of the intermediates. In this way a 70–75% yield of 3-O-benzyl-sn-glycerol with 94–96% ee has been obtained.