A bioprocess utilising the extracellularly producing biosurfactant, Candida bombicola ATCC 2214 for the uptake of palm oil as secondary substrate has been investigated. The biosynthesis of extracellular sophorolipid biosurfactant, constituting of a 26 membered lactone ring and free acid open-ring compounds. is associated with microbial growth on lipid and glucose as substrates. The main objective of this research project is to optimise the production of sophorolipid biosurfactant by utilisation of various types of palm oil as substrate by studying the bioprocess factors affecting the yields and efficiencies. In addition, we have investigated the biological activity of the sophorolipid against microorganisms and studied the application of MALDI-ToF MS technique in the rapid characterisation of sophorolipid and palm oil. High yield of sophorolipid was obtained when Candida bambicola ATCC 22214 was grown on a mixture of glucose and palm olein. Process optimisation studies for the production of the biosurfactant was initiated in shake flask cultures and further investigation were conducted in laboratory scale bioreactors. The product yield increased to 168 g/L sophorolipid with the addition of 0.5% yeast extract to the cultivation medium in a 5 L batch bioreactor. The recovered sophorolipid showed good surface active properties and antimicrobial activity. Sophorolipid at the concentration of 450 mglL reduced the surface tension of water to 34.5 mN/m. The sophorolipids demonstrated significant antimicrobial activity against actinomycetes and other microbial species. In this work, we report the first research studies using MALDI-ToF Mass Spectrometry as an analytical tool to detect and characterise sophorolipids using THAP, DHB and 3•hydroxy picolinic acid as matrices. 3-hydroxy picolinic acid produced superior results compared to the two other matrices. Peaks corresponding to the 26membered lactonic ring and O-acetylated forms of sophorolipids were clearly visualised. Protocols for semi-quantitative MALDI-ToF MS measurements of the various substructures of the sophorolipids were developed. In the positive ion acquisition mode the data reveals that the Glc-Glc-1'-,4"-C1B:1 component account for approximately 7.1% of the total molecular ions observed and the neutral component of the GlcOAc-Glc-1'-,4"-C18:1 ion at 2.7%. The acidic form of the GlcOAC-Glc-1'-C18:1 molecular ion has 3.1% relative intensity while the peaks representing neutral GlcOAcGlcOAc- 1'-,4"-C18:1 fragment and its anionic cognate has relative intensity values of 12 and 7.5 % respectively. The MALDI-ToF Mass Spectrometry technique developed in this study provided a very rapid and high sensitive method for the analysis of a complex biomolecular mixture of sophorolipids without the need for any prior chromatographic separation processes.