High-molecular-weight (HMW) surface-active agents (biosurfactants and bioemulsifiers) are produced by many different types of bacteria and comprise proteins, glycoproteins, lipoproteins, polysaccharides, lipopolysaccharides or complexes containing any combination of these structural types. The attraction of HMW glycoprotein bioemulsifiers as ingredients in food and drink formulations, for example, has increased in recent years, especially those produced by marine bacteria, as these amphiphilic macromolecules can offer improved emulsifying and emulsion stability properties. Marine bacteria are a largely untapped source for these types of molecules, and unlike those produced by terrestrial bacteria and microalgae, marine bacterial glycoproteins are often highly negatively charged (polyanionic) which endows these macromolecules with potential multifunctional properties. The polyanionic nature and molecular-weight heterogeneity of these types of molecules require careful attention to optimising their isolation from complex media. This chapter provides a detailed description for optimising the isolation/separation of amphiphilic polyanionic glycoprotein bioemulsifiers produced by marine bacteria.