Lignin is a biomass-derived aromatic polymer that has been identified as a potential renewable source of aromatic chemicals and other valuable compounds. The valorization of lignin, however, represents a great challenge due to its high inherent functionalization, what compromises the identification of chemical routes for its selective depolymerization. In this work, an in vitro biocatalytic depolymerization process is presented, that was applied to lignin samples obtained from beech wood through OrganoCat pretreatment, resulting in a mixture of lignin-derived aromatic monomers. The reported biocracking route comprises first a laccase-mediator system to specifically oxidize the C alpha hydroxyl group in the beta-O-4 structure of lignin. Subsequently, selective beta-O-4 ether cleavage of the oxidized beta-O-4 linkages is achieved with beta-etherases and a glutathione lyase. The combined enzymatic approach yielded an oily fraction of low-molecular-mass aromatic compounds, comprising coniferylaldehyde and other guaiacyl and syringyl units, as well as some larger (soluble) fractions. Upon further optimization, the reported biocatalytic route may open a valuable approach for lignin processing and valorization under mild reaction conditions.