Biosurfactants offer good advantages over synthetic counterparts, including biodegradability, environmentally friendly and low toxicity. This study employed a yeast Meyerozyma guilliermondii MX strain for bioconversion of lignocellulosic xylose and palm oil to valuable glycolipid biosurfactant with desirable properties. The objective was to elucidate metabolic pathways related to production of glycolipids and its functional properties. To enhance de novo glycolipid production, manipulation of responsible enzymatic genes was conducted using media and environmental means in comparison to the industrial glycolipid producer, Candida bombicola. Proteomic profiles of yeast cells grown with or without palm oil uncovered novel key metabolic enzymes, namely fatty acid biosynthetic enzymes, leading to formation of glycolipid precursors. qRT-PCR identified some cluster genes responsible for biosynthesis of desirable glycolipids. Finally, LC-MS-based lipidomics of glycolipid fraction identified 15-(2'-O-β-D-glucopyranosyl-β-D-glucopyranosyloxy)hexadecanoic acid 1',4″-lactone 6',6″-diacetate (663.4525 m/z) as a major product. Using co-carbon substrates in the presence of salt and zinc, maximum glycolipid yield was achieved (55.72 g/L) with 55.30% emulsification activity and 10 mg/L of CMCs. Mixed glycolipids demonstrated antibiofilm activity against Candida albicans shown by reduction of metabolic activity. The novel biosurfactant-producing yeast M. guilliermondii MX is a promising cell factory of new antibiofilm glycolipids with potential for industrial-scale up.