Lignin is a biopolymer found abundantly in nature which is a promising sustainable feedstock for biotechnological applications, due to its carbon content and aromatic nature. However, lignin’s resistance to chemical and microbial degradation has limited its use. Some accessory enzymes have been involved in lignin degradation. Among these enzymes are copper-containing glyoxal oxidases from white-rot fungi (Phanerochaete Chrysosporium), which catalyse the oxidation of aldehydes to their corresponding carboxylic acids. In this study, a flavin-dependent enzyme annotated as glycolate oxidase and encoded by gene ro02984 in Rhodococcus jostii RHA1 previously shown to oxidise mandelic acid to phenylglyoxalic acid, is investigated as a possible accessory enzyme for lignin degradation. The recombinant enzyme was purified by Immobilized Metal Affinity Chromatography (IMAC). The subunit molecular weight of glycolate oxidase was determined by SDS-PAGE to be 59 kDa. The steady state kinetic parameters have been elucidated for this enzyme with glyoxal, methylglyoxal, lactic acid, malic acid, 4-hydroxphenylglyoxal, glycolic acid and glycolaldehyde. The products of these oxidations have been determined. The results show that glycolate oxidase enzyme has the ability to oxidise α-hydroxy acid, phenylglyoxal, and aldehyde groups. Furthermore, glycolate oxidase catalyses each of the steps from glycolaldehyde to oxalic acid, and this provides a possible pathway for the formation of oxalic acid from lignin which was proved using labelled DHP lignin. In addition, we have demonstrated that R. jostii glycolate oxidase acts as an efficient coupling enzyme for two bacterial Dyp peroxidases, by generating the hydrogen peroxide co-substrate needed by the peroxidase enzyme. Furthermore, the ability of glycolate oxidases to oxidise HMF to its valuable derivatives was shown in this study.