An extensive amount of research has been conducted on a multitude of emulsifiers regarding their effect on o/w emulsion characteristics and lipolysis kinetics. However, there is an emerging need towards the understanding of biobased emulsifier characteristics. Therefore, this research studied the effect of 6 glycolipids on interfacial tension, emulsion microstructure throughout in vitro digestion, and lipolysis kinetics. Findings showed that molecular differences between glycolipids, such as the degree of acetylation, lactonization, and symmetry, substantially affected their behavior on the oil-water (o/w) interface, lowering the interfacial tensions to values ranging between 2 and 18 mN/m. Glycolipids with a higher amount of acetyl groups, lower tendency to self-assemble, and/or smaller molecular volume on the interface, decreased the interfacial tension substantially more. Therefore, acetylated lactonic sophorolipid decreased the interfacial tension most, while non-acetylated sophoroside showed the smallest effect on the interfacial tension. While all emulsions were stable and initially had similar droplet sizes, some were unstable throughout the simulated upper digestive tract, resulting in significantly different hydrolysis behaviors. Acetylated lactonic sophorolipid and non-acetylated glucolipid were more hydrophobic than the remaining 4 glycolipids, causing this gastric instability resulting in lower lipolysis extents by the end of the small intestinal phase. The acetylated sophoroside emulsion was unstable during the small intestinal phase, attributed to bile salt interactions. Therefore, this research concludes that molecular changes between glycolipids give rise to significantly different emulsion and digestion properties. These insights can be used in future work to optimize glycolipid structure and subsequent functional properties.