Mannosylerythritol lipids (MELs) are glycolipid biosurfactants with versatile applications in green chemistry, personal care and bioremediation, owing to their excellent surface activity and biosustainability. Despite their potential, a direct comparative evaluation of MEL production by Pseudozyma antarctica and Ustilago maydis using a sustainable carbon source such as palm olein has not been reported. This study addresses this gap by cultivating both strains under optimised submerged fermentation conditions over 10 days to assess species-dependent differences in yield and product characteristics. The MELs were isolated by solvent extraction and further purified by column chromatography. A comprehensive characterisation of the biosurfactants was performed using surface tension measurements, liquid chromatography-mass spectrometry (LC-MS), Fourier transform infrared spectroscopy (FTIR), and nuclear magnetic resonance spectroscopy (NMR). The results showed that P. antarctica produced a significantly higher MELs yield (58.37 g/L) compared to U. maydis (26.77 g/L). The purified MELs showed a critical micelle concentration (CMC) of 2.8 and 2.6 mg/mL, respectively, confirming their strong surfactant properties. The LC-MS and NMR analyses tentatively confirmed the presence of MEL homologs with fatty acid chains from C6 to C20. These results emphasise the feasibility of using palm olein as an effective and renewable substrate for MELs production and establish their applicability as sustainable, bio-based alternatives to conventional synthetic surfactants.